Name: free radicals in chemical biology: from chemical behavior to biomarker development
Uploaded: 2013-04-15T13:00:00
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Description: Watch this Scientific Journal Video about Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development at JoVE.com

Financial support from the Ministero dell'Istruzione, dell'Universit&aacute; della Ricerca (PRIN-2009K3RH7N_002) and Marie Curie Intra-European Fellowship (CYCLOGUO-298555) as well as the sponsorship of COST Action CM0603 on 'Free Radicals in Chemical Biology and COST Action CM1201 on "Biomimetic Radical Chemistry" are gratefully acknowledged.

1. Synthesis of Mono-trans Isomers of Cholesteryl Esters
<ol>
 <li>Dissolve cholesteryl esters (linoleate or arachidonate cholesteryl ester) in 2-propanol (15 mM) . For a better solubilization, the samples were sonicated for 15 min under argon.</li>
 <li>Transfer the solution to a quartz photochemical reactor, add 2-mercaptoethanol in 2-propanol (to reach 7 mM concentration, from a 2 M stock solution of the thiol). Flush the reaction mixture with argon for 20 min in order to eliminate the presence of oxygen in the solution.</li>
 <li>Irradiate the reaction mixture by UV light using a 5.5W low-pressure mercury lamp at 22&plusmn;2 &deg;C for 4 min. Monitor by analytical Ag-TLC (silver-thin layer chromatography) to evidence the formation of the mono-trans cholesteryl esters (see Figure 1 for the chemical formulas). The TLC staining is carried out by pouring the plate in the cerium ammonium molibdate (CAM) solution, and the spots appear on heating the plate. Quench the reaction at an early stage, in order to recover the starting material, that can be reused to perform other rounds of isomerization, obtaining an increase of the overall yield.</li>
 <li>Collect the reaction mixture in a round-bottom flask, washing the apparatus with a few ml of 2-propanol. Remove solvent and purify the mono-trans isomers of cholesteryl esters by Ag-TLC as described in the literature.5 Use hexane-diethyl ether (9:1 v/v) as the eluent for mono-trans cholesteryl linoleate isomers, whereas use hexane-diethyl ether-acetic acid (9:1:0,1 v/v) for mono-trans cholesteryl arachidonate isomers.</li>
</ol>
2. Isolation of Cholesteryl Esters Fraction from Human Serum
<ol>
 <li>Dilute 1 ml of human serum (obtained by centrifugation of blood) with 1 ml of brine and pour the solution into a separatory funnel under a stream of argon in order to avoid artifacts (e.g. oxidation adducts). Add 10 ml chloroform-methanol (2:1 v/v) thrice. Shake the separatory funnel very slowly in order to limit the formation of the emulsion due to the presence of albumin.</li>
 <li>Wash the organic layers once with brine (10 ml), then collect in an Erlenmeyer flask under an argon flow, dry over anhydrous sodium sulphate. Remove volatiles by rotary evaporator to afford a yellow oil (total plasma lipid fraction).</li>
 <li>Take up the crude with 1 ml of chloroform-methanol (2:1 v/v), load onto a preparative TLC under a stream of argon and use a mixture of hexane-diethyl ether (9:1 v/v) as the eluent. Scrape off the silica portion containing the cholesteryl ester fraction and pour into a vial. Then, extract silica (3 &times; 5 ml) with chloroform-methanol (2:1 v/v), collect the organic layers and remove the solvent after evaporation to afford the pure fraction of cholesteryl esters (usually ~1.5 mg).</li>
 <li>Keep cholesteryl esters in a dark vial covered by aluminum foil under Argon and store at -20 &deg;C. Cholesteryl esters are sensitive to light and oxygen.</li>
</ol>
3. Characterization of Mono-trans Cholesteryl Esters by Raman Spectroscopy
<ol>
 <li>Extract cholesteryl esters from human serum (&ge;0.7 mg) as described in section 2, dissolve in a small amount of carbon tetrachloride (&le;10 &mu;l) and place in a vial. CCl4 is selected as the solvent because its Raman signal does not overlap with the region of interest of cholesteryl ester analysis.</li>
 <li>Transfer the solution to the sample holder through a disposable glass pipette, then carefully remove the solvent by a slow stream of argon. Once a uniform oily film is formed on the internal wall of the sample holder, place the latter into the instrument for measurement.</li>
 <li>Fourier Transformed Raman spectrum of cholesteryl esters is directly obtained on the lipid extracts without any derivatization reaction. The laser power on the sample is &lt;100 mW to avoid sample damage. The total number of scans for each spectrum is &ge;800 to minimize the background noise.</li>
 <li>The 1,700-1,630 cm-1 range of the Raman spectrum is analysed since it reflects the contribution from the C=C double bonds present in the molecule (C=C stretching mode). A curve fitting analysis is performed on this region, thus allowing the distinguishing of the superimposed contributions from cis and trans double bonds in the fatty alkyl chain and double bond in the ring B of cholesterol (see Figure 2).</li>
 <li>To fit correctly the vibrational bands, some parameters should be fixed or constrained within reasonable limits. The component peak profiles are described as a linear combination of Lorentzian and Gaussian functions, whereas the half-height bandwidths are best determined by the computer optimisation routine. The number and position of the component peaks are obtained by using the fourth derivative spectra, which allow to detect also some weak component bands. Since the signal to noise ratio which deteriorates upon differentiation of the signal can preclude the use of the fourth derivative, smoothed fourth derivatives with a thirteen-point Savitsky-Golay function, are of advantage; thus, a right compromise between resolution and signal to noise ratio is obtained. Best curve fitting are obtained at the lowest possible c2 values.</li>
 <li>The presence of trans isomers is revealed by the component at 1671&plusmn;1 cm-1, in addition to, at least, the two components, slightly shifted towards lower frequencies, due to the fatty acid chain and cholesterol C=C double bonds. Representative Raman spectrum of plasma cholesteryl ester is shown in Figure 9. In the inset the comparison of a specific region with cholesteryl linoleate and mono-trans cholesteryl linoleic acid isomers is shown.</li>
</ol>
4. Derivatization to Fatty Acid Methyl Esters (FAME) and Analysis by Gas Chromatography
<ol>
 <li>Dissolve cholesteryl esters (~1.5 mg) with 0.5 ml of a 1 M solution of sodium hydroxide in benzene-methanol (2:3 v/v) and place in a dark vial covered by aluminum foil under argon.</li>
 <li>Leave the mixture stirring at room temperature and monitor by TLC using a mixture of hexane-diethyl ether (9:1 v/v) as the eluent. Reaction time is usually 30 min but a careful reaction monitoring is required. In fact, once the corresponding methyl esters are formed, the reaction must be quenched in order to avoid the formation of degradation and side products. TLC showed the quantitative formation of methyl esters.</li>
 <li>Quench the reaction mixture by adding 1 ml of brine and extract methyl esters with n-hexane (3 &times; 2 ml). Collect the organic layers into a vial and remove the solvent by rotary evaporation to afford the methyl ester fraction, which is then used for GC analysis without further purification.</li>
 <li>Dissolve the methyl esters in the minimum quantity of n-hexane (usually 1 mg in 50 &mu;l) and inject in the GC equipped with a 60 m &times; 0.25 mm &times; 0.25 &mu;m (50%-cyanopropyl)-methylpolysiloxane column. Use a flame ionization detector (FID) with the following oven program: temperature start at 165 &deg;C, hold for 3 min, followed by increase of 1 &deg;C/min up to 195 &deg;C, hold for 40 min, followed by a second increase of 10 &deg;C/min up to 240 &deg;C, and hold for 10 min. A constant pressure mode (29 psi) is chosen. Helium or hydrogen can be used as the carrier gas. Methyl esters are identified by comparison with the retention times of authentic samples. Figure 10 shows a representative GC analysis of FAME obtained from plasma cholesteryl esters.</li>
</ol>
5. Synthesis of (5'R)- and (5'S)-5',8-cyclo-2'-deoxyadenosine
<ol>
 <li>Dissolve 33 mg of 8-bromo-2'-deoxyadenosine (8-Br-dAdo) in acetonitrile (100 ml) in order to reach 1 mM concentration. Regulate gas flow (Ar or N2) in the photoreactor and fill the apparatus with the solution of 8-Br-dAdo.</li>
 <li>Prepare a septum with the appropriate size for the photoreactor's input and pass a needle connected to the inert gas line through the center of it. Close the system with the septum. Flush the inert gas for 30 min.</li>
 <li>Irradiate by UV light using a 125W medium-pressure mercury lamp at 22&plusmn;2 &deg;C for 15 min, collect the solution into a 250 ml round-bottomed flask. Wash the photoreactor with 10 ml acetonitrile and collect washings in the same flask.</li>
 <li>Quench the crude reaction mixture with 1 M NH4OH solution and evaporate the solvent in the rotary evaporator.</li>
 <li>Run high performance liquid chromatographic analysis (HPLC-UV) with analytical column (see instrumentation section) under known conditions , and compare the profile with reference compounds.</li>
 <li>Run high performance liquid chromatographic analysis (HPLC-UV) with an analytical column (see instrumentation section) by using the following solvents and gradients: 2 mM ammonium formate as solvent A and acetonitrile as solvent B. Set the flow rate to 1 ml/min and the gradient from 0% solvent B to 0.3% solvent B to be reached in 2.2 min. Then 0.8% solvent B in in 4.0 min, then 1% solvent B in 4.8 min. Remain at 1% solvent B for 9 min and then go to 8% solvent B in 6 min. After go to 10 % solvent B in 4 min and to 30% solvent B in 5 min and remain at 30% solvent b for other 5 min. Collect the chromatographic peaks corresponding to the two diastereomeric products in two different vials.</li>
 <li>Measure the absorbance of the two collected fractions in the UV spectrophotometer and calculate the exact concentration based on the Lamber-Beer law (A = &epsilon;lC, where A is the absorbance, &epsilon; the extinction coefficient and l the length of the cell). Use the extinction coefficient &epsilon; of 2'-deoxyadenosine (dAdo) (15,400 M-1 cm-1 at 260 nm).</li>
</ol>
6. Synthesis of (5'R)- and (5'S)-5',8-cyclo-2'-deoxyguanosine
<ol>
 <li>Dissolve 35 mg of 8-bromo-2'-deoxyguanosine (8-Br-dGuo) and 30 mg of sodium iodide (NaI) in distilled water (100 ml) in order to reach 1mM and 2mM concentration, respectively. Regulate inert gas (Ar or N2) flow connected to the photoreactor and fill the photoreactor with the reaction solution.</li>
 <li>Degass the reaction mixture as described in procedure 5 (step b).</li>
 <li>Irradiate by UV light using a 125W medium-pressure mercury lamp at 22&plusmn;2 &deg;C for 30 min, collect the solution into a 250 ml round-bottomed flask. Wash the photoreactor with 10 ml of water and collect washings in the same flask.</li>
 <li>Quench the crude reaction mixture with 1 M NH4OH solution and evaporate the solvent under vacuum.</li>
 <li>Perform analysis as described in Procedure 5 (steps e to g). Use the extinction coefficient &epsilon; of 2'-deoxyguanosine (dGuo) (11,700 M-1 cm-1 at 260 nm).</li>
</ol>
7. Synthesis of Isotopic Labeled Purine (5'R)- and (5'S)-5',8-cyclonucleosides
<ol>
 <li>Prepare 2 ml of 1mM aqueous solution (distilled water) of the 15N isotopic labeled purine nucleosides ([15N5]dAdo or [15N5]dGuo) in a glass vial (4 ml) with a septum cap.</li>
 <li>Connect the vial with the septum cap and connect to the gas line (N2O for saturation of the solution) through a needle in the septum that reaches the bottom of the vial. Another short needle in the septum cap works as the gas exit.</li>
 <li>Flush the solution with N2O for 30 min. The gas flow is regulated to be very low.</li>
 <li>The exit needle is first taken out and after 1' the long one follows, in order to have a small pressure inside the vial.</li>
 <li>Put the solution in the apparatus of gamma radiolysis for 8 hr (calculation on the basis of a dose rate ca. 4.5 Gy/min).</li>
 <li>After reaction quench the crude with 1 M NH4OH solution.</li>
 <li>Run high performance liquid chromatographic analysis (HPLC-UV) under known conditions,6 and compare the chromatogram with the standard references.</li>
</ol>
8. Gamma Radiolysis of DNA Aqueous Solutions
<ol>
 <li>Prepare 1 ml of 0.5 mg/ml solution (distilled water) of calf thymus DNA and put in a glass vial (2 ml) with a septum cap.</li>
 <li>Degass the reaction as described in Procedure 7 (steps b-d).</li>
 <li>Put the solution in the apparatus of gamma radiolysis for 30 min (calculation on the basis of dose/rate ca. 4.5 Gy/min).</li>
</ol>
9. Enzymatic Digestion of DNA
<ol>
 <li>To an Eppendorf tube containing 80 &mu;g of DNA, add 8 U of nuclease P1, 0.01 U of phosphodiesterase II, 20 nmol of EHNA in 20 &mu;l of 300 mM sodium acetate (pH 5.6) and 10 mM zinc chloride. Incubate the mixture at 37&deg;C for 48 hr.</li>
 <li>Next add a mixture of 8 U of alkaline phosphatase, 0.02 U of phosphodiesterase I, in 40 &mu;l of 0.5 M Tris-HCl buffer (pH 8.9) to mixture digestion. The sample is incubated at 37&deg;C for 2 hr.</li>
 <li>The mixture is neutralized by addition of 10% formic acid.</li>
 <li>Transfer the sample to an Amicon Ultra-0.5 centrifugal filter device (cutoff 3 kDa), add 200 &mu;l of tridistilled H2O. Place the Ultra-filter into the centrifuge rotor at 14,000 x g for 15 min. Then separate the Ultra filter device from the microcentrifuge tube. Lyophilize the enzyme-free solution in the microcentrifuge tube.</li>
</ol>
10. DNA Sample Desalinization Prior to Analysis
<ol>
 <li>Run high performance liquid chromatographic analysis (HPLC-UV) with analytical column (see instrumentation section) following known conditions.6</li>
 <li>Dissolve the lyophilized digested DNA in 20 &mu;l of H2O and inject in HPLC-UV.</li>
 <li>Collect the sample in a vial after the salt elution (first minutes) and right before the elution time of the first nucleoside (5'R-cdGuo), until the end of the chromatographic program.</li>
 <li>The sample is lyophilized and resolubilized in 20 &mu;l water.</li>
</ol>
11. LC-MS/MS Quantitative Analysis
<ol>
 <li>Prepare the HPLC-MS/MS (triple quadrupole) before starting the analysis by loading the analytical method and the method for the MS detector (ESI).6</li>
 <li>Add 1 &mu;l of the isotopic labelled compounds mixture in the sample prepared in Procedure 7.</li>
 <li>Inject 20 &mu;l of the spiked digested DNA solution in distilled water.</li>
 <li>The obtained chromatographic data are elaborated on the basis of previous calibration with reference compounds. A representative HPLC run containing adenosine and guanosine 2'-deoxyribonucleosides and their oxidative and cyclo derivatives is shown in Figure 11.</li>
</ol>

<ol>
	<li>Ferreri, C., Chatgilialoglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipidomics+and+the+geometry+of+unsaturated+fatty+acids:+from+biomimetic+models+to+biological+consequences.">Membrane lipidomics and the geometry of unsaturated fatty acids: from biomimetic models to biological consequences.</a> Methods Molecular Biology. 579, 391-412 (2009).</li><li>Chatgilialoglu, C., Ferreri, C., Terzidis, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purine+5',8-cyclonucleoside+lesions:+chemistry+and+biology.">Purine 5',8-cyclonucleoside lesions: chemistry and biology.</a> Chemical Society Reviews. 40 (3), 1368-1382 (2011).</li><li>Chatgilialoglu, C., Ferreri, C., 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=Radiation-induced+reductive+modifications+of+sulfur-containing+amino+acids+within+peptides+and+proteins.">Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins.</a> Journal of Proteomics. 74 (11), 2264-2273 (2011).</li><li>Chatgilialoglu, C., Ferreri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans+lipids:+the+free+radical+path.">Trans lipids: the free radical path.</a> Accounts Chemical Research. 38 (6), 441-448 (2005).</li><li>Melchiorre, M., Torreggiani, A., Chatgilialoglu, C., Ferreri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+markers+of+geometrical+radical+stress:+synthesis+of+mono-trans+cholesteryl+esters+isomers+and+detection+in+human+plasma.">Lipid markers of geometrical radical stress: synthesis of mono-trans cholesteryl esters isomers and detection in human plasma.</a> Journal of the American Chemical Society. 133 (38), 15184-15190 (2011).</li><li>Wang, J., Yuan, B., Guerrero, C., Bahde, R., Gupta, S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Oxidative+DNA+Lesions+in+Tissues+of+Long-Evans+Cinnamon+Rats+by+Capillary+High-Performance+Liquid+Chromatography-Tandem+Mass+Spectrometry+Coupled+with+Stable+Isotope-Dilution+Method.">Quantification of Oxidative DNA Lesions in Tissues of Long-Evans Cinnamon Rats by Capillary High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method.</a> Analytical Chemistry. 83 (6), 2201-2209 (2011).</li><li>Boussicault, F., Kaloudis, P., Caminal, C., Mulazzani, Q. G., Chatgilialoglu, 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+fate+of+C5'+radicals+of+purine+nucleosides+under+oxidative+conditions.">The fate of C5' radicals of purine nucleosides under oxidative conditions.</a> Journal of the American Chemical Society. 130 (26), 8377-8385 (2008).</li><li>Ferreri, C., Kratzsch, S., Brede, O., Marciniak, B., Chatgilialoglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans+lipids+formation+induced+by+thiols+in+human+monocytic+leukemia+cells.">Trans lipids formation induced by thiols in human monocytic leukemia cells.</a> Free Radicals Biology &amp; Medicine. 38 (9), 1180-1187 (2005).</li><li>Zambonin, L., Ferreri, C., 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=Occurrence+of+trans+fatty+acids+in+rats+fed+a+trans-free+diet:+a+free+radical-mediated+formation.">Occurrence of trans fatty acids in rats fed a trans-free diet: a free radical-mediated formation.</a> Free Radicals Biology &amp; Medicine. 40 (9), 1549-1556 (2006).</li><li>Puca, A. A., Andrew, P., 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=Fatty+acids+profile+of+erythrocyte+membranes+as+possible+biomarker+of+longevity.">Fatty acids profile of erythrocyte membranes as possible biomarker of longevity.</a> Rejuvenation Research. 11 (1), 63-72 (2008).</li><li>Belmadoui, N., Boussicault, F., 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=Radiation-induced+formation+of+purine+5',8-cyclonucleosides+in+isolated+and+cellular+DNA:+high+stereospecificity+and+modulating+effect+of+oxygen.">Radiation-induced formation of purine 5',8-cyclonucleosides in isolated and cellular DNA: high stereospecificity and modulating effect of oxygen.</a> Organic &amp; Biomolecular Chemistry. 8 (14), 3211-3219 (2010).</li><li>Chatgilialoglu, C., Bazzanini, R., Jimenez, L. B., Miranda, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(5'S)-+and+(5'R)-5',8-cyclo-2'-deoxyguanosine:+mechanistic+insights+on+the+2'-deoxyguanosin-5'-yl+radical+cyclization.">(5'S)- and (5'R)-5',8-cyclo-2'-deoxyguanosine: mechanistic insights on the 2'-deoxyguanosin-5'-yl radical cyclization.</a> Chemical Research in Toxicology. 20 (12), 1820-1824 (2007).</li><li>Wang, J., Clauson, C. L., Robbins, P. D., Niedernhofer, L. J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oxidative+DNA+lesions+8,5'-cyclopurines+accumulate+with+aging+in+a+tissue-specific+manner.">The oxidative DNA lesions 8,5'-cyclopurines accumulate with aging in a tissue-specific manner.</a> Aging Cell. 11 (4), 714-716 (2012).</li><li>Ferreri, C., Chatgilialoglu, 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+role+of+fatty+acid-based+functional+lipidomics+in+the+development+of+molecular+diagnostic+tools.">The role of fatty acid-based functional lipidomics in the development of molecular diagnostic tools.</a> Expert Review of Molecular Diagnostics. 12 (7), 767-780 (2012).</li></ol>

No conflicts of interest declared.

The conversion of naturally occurring cis unsaturated fatty acids to geometrical trans isomers is a transformation connected with the production of radical stress in the biological environment. Cell membrane lipids, which contain fatty acids, are a relevant biological target for radical stress and we first studied the endogenous cis-trans phospholipids isomerization in cell cultures, animals and humans assessing analytical protocols in each case.8-10 We demonstrated that this transformation can occur by a variety of S-containing compounds, including thiols, thioethers and disulfides, which under different radical stress conditions are able to generate thiyl radicals, i.e. the isomerizing agent (Figure 3). The example shown in this article focuses on the class of cholesteryl esters, which represent a well-known fraction of plasma lipids, strictly involved in lipoprotein metabolism. The ester linkage between fatty acids and cholesterol is biosynthesized by the transfer of fatty acids from the position 2 of the glycerol moiety of phosphatidylcholine to cholesterol, a step catalyzed by the enzyme lecithin cholesterol acyl transferase (LCAT). Therefore, plasma cholesteryl esters are strictly connected with membrane lipid turnover, and contain relatively high proportions of the polyunsaturated fatty acids (PUFA) typically present in phosphatidylcholines, i.e. linoleic and arachidonic acids. Lipoprotein formation is involved in cardiovascular and metabolic diseases. The reactivity of natural cholesteryl esters with free radicals can occur at the double bonds of linoleate and arachidonate residues, which can be transformed in the corresponding trans geometrical isomers (see Figure 1 for the structures). Characterization of the trans cholesteryl ester content in biological samples is interesting for biomarker development. An indirect methodology consists of the transformation of cholesteryl esters isolated from plasma to the corresponding fatty acid methyl esters (FAME) and separation by gas chromatographic protocols. In this case, the calibration of the standard references of cis and trans fatty acid methyl esters is performed, in order to allow the quantitation of the trans content in the samples. Based on the analytical studies carried out on the cholesteryl ester library, we proposed to apply also a method based on Raman spectroscopy, that can be carried out directly on the cholesteryl ester fraction isolated from plasma, without further derivatization to the corresponding FAME (see Figures 2 and 9). It is worth noting that up to now no successful methods are described to separate cis and trans isomers of fatty acid containing lipids by HPLC, as instead described by cholesteryl ester hydroperoxides. So far, the indirect gas chromatographic method is still the best available method so far. By this method the first quantitative evaluation of the mono-trans content derived from cholesteryl esters isolated from plasma of healthy subjects was provided. Using Flame Ionization Detector (FID) the limit of detectability is satisfactory (ppb) and nanomolar quantities of the compounds have been detected.5. With different detection systems this limit can be even lowered. The effect of ionizing radiation on cholesteryl esters is matter of further studies, whether a linear response is obtained relative to the applied dose.
As a second example, we chose modified nucleosides which can be produced by free radical damage of DNA. Hydroxyl radicals (HO&bull; ) are known to be the most harmful Reactive Oxygen Species (ROS) for their ability to cause chemical modifications to DNA. Single or multiple lesions may occur on DNA, that in eukaryotic cells is located in nucleus and mitochondria. Identification and measurement of the main classes of oxidative generated damages to DNA require the appropriate molecular libraries in order to set up the analytical protocols. We focused our interest on the smallest tandem lesions, which are purine 5',8-cyclo-2'-deoxyribonucleosides, having an additional covalent bond between the base and the sugar moieties created by the free radical attack. The compounds are 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine existing in the 5'R and 5'S diastereomeric forms (Figure 5). Their potential to become free radical stress marker is matter of fundamental research.2 Indeed, when DNA is exposed to HO&bull; radical hydrogen abstraction from C5' position of the sugar is one of the possible events leading to the formation of these tandem lesions. Purine 5&#x2032;,8-cyclonucleosides can be measured as sum of diastereomers by HPLC-MS/MS in enzymatically digested &gamma;-irradiated DNA samples varying from 1 to 12 lesions /106 nucleosides/Gy going form absence of oxygen to physiological level of oxygen in tissues, the diastereomeric ratio 5'R/5'S being ~4 and ~3 for 5',8-cdAdo and 5',8-cdGuo, respectively (Figure 11).11 It is worth noting that the relationship between radiation dose and 5',8-cdAdo and 5',8-cdAdo lesions detected in cellular DNA is far from being understood. The single experiment based on 2kGy irradiation reported in the experimental cannot be considered conclusive.11 Further experiments of this kind and analytical quantitation of the four lesions are necessary for defining such relationship. The detection of these lesions and the more popular oxidative transformations (such as 8-oxo-2'-deoxyguanosine, 8-oxodGuo) are matter of intense investigations, evidencing the importance of both lesions during oxidative metabolism.6,13 The use of HPLC-MS/MS (triple quadrupole) has a detection limit close to 30fmol for all four lesions. Last improvements are claimed to reach detection limits of attomol levels by the instrument producers. Based on the very recent literature,6 analytical procedures have to include the proper cleanup of the sample and enrichment in order to meet the detection limits of the MS/MS/MS (ion trap) or of the MS/MS (triple quadrupole) used in our case.
Bio-inspired synthetic procedures of compounds 1-4 were developed starting from 8-bromopurine derivatives under or photolysis.7,12 These procedures involve a radical cascade reaction that mimics the DNA damage mechanism of formation of 5',8-cdAdo and 5',8-cdGuo lesions. From biological perspectives, it was found that these lesions accumulate with aging in a tissue-specific manner (liver>kidney>brain), providing evidence that DNA repair mechanisms are inadequate to preserve the genetic material from these lesions.13 Indeed, nucleotide excision repair (NER) is the only pathway currently identified for the repair of these lesions.2 
The two classes of compounds shown in Figures 1 and 5 are not commercially available at the moment, however by the synthetic strategies described in the literature it would not be difficult to prepare these compounds for commercial use.
The multidisciplinary approach provided by chemical biology studies not only has an enormous value in the identification of novel mechanisms occurring in the biological environment, but also gives a fundamental contribution to biomarker discovery and diagnostics, ultimately bringing novelty in health care and prevention strategies.14 The chemical contribution is needed for a successful development of molecular medicine, creating integrated platforms and panels for metabolic profiling which are expected to allow for an optimal rationalization of intervention design, either therapeutic and nutritional, reducing uncertainties and failures when they can be predictable.

The reactivity of free radicals revealed its enormous importance for many biological events, including aging and inflammation. Nowadays, it is more and more evident that the clarification of each chemical step involved in this reactivity is needed, in order to understand the underlying mechanisms and envisage effective strategies for the control of free radicals and repair of the damage. The contribution from chemical studies is fundamental, but the direct study in the biological environment can be difficult, since the superimposition of different processes complicates and perturbs the examination of the results and the related conclusions. Therefore, the strategy of modeling free radical reactions under biologically related conditions has become a fundamental step in the research of chemical mechanisms in biology. 
In the last decade our group developed models of free radical processes under biomimetic conditions. In particular we envisaged biologically relevant transformations of unsaturated fatty acids, nucleosides, and sulfur-containing amino acids and put them in the track to be evaluated and validated as biomarkers of health status.1-4 
Our general approach consists of three modules:
<ul>
 <li> 	Organic synthesis, which provides access to appropriately modified biomolecules. The synthetic plan can be also designed in order to simulate the free radical processes occurring in the biological environment, thus applying conditions able to simulate the biological process of interest, which is the principle of biomimetic radical chemistry. In biomimetic models the reaction medium is water or a heterogeneous medium due to the coexistence of hydrophilic and hydrophobic compartments. The advantage to work with biomimetic models is to have a simplified environment with known reaction partners, where reactivity and products can be examined in more details, possibly discovering novel chemical pathways. From this step mechanistic and kinetic information can be gathered; </li>
 <li> 	Purification, analysis and characterization of the products, providing structural and chemical information of the modified biomolecules in order to organize molecular libraries and facilitate recognition.in the more complex biological environment. Protocols are established also in view of resolving complex mixtures of compounds such as those derived from biological specimens; </li>
 <li> 	Biomarker development using biological samples, derived either by in vitro experiments using cell cultures, and in vivo studies from animals and humans. The protocols developed in biomimetic models are then applied to the complex analysis of samples, to envisage the free radical transformations under different conditions. Molecular libraries developed by synthesis are of enormous help to life science discoveries. The information gathered on the free radical transformations give the opportunity to figure out repair and prevention strategies. Databases of the results can be used for a careful evaluation by multivariate analysis of the biomarker significance as well as possible associated factors. </li>
</ul>
We chose two classes of relevant biomarkers to accredit this approach: cholesteryl esters and purine 5',8-cyclo-2'-deoxyribonucleosides.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>MATERIALS</td>
 </tr>
 <tr>
 <td>Cholesteryl linoleate &ge;98%</td>
 <td>Sigma-Aldrich</td>
 <td>C0289-100 mg</td>
 <td></td>
 </tr>
 <tr>
 <td>Cholesteryl arachidonate&ge;95% </td>
 <td>Sigma-Aldrich</td>
 <td>C8753-25mg</td>
 <td></td>
 </tr>
 <tr>
 <td> 2-mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M6250-100 ml</td>
 <td></td>
 </tr>
 <tr>
 <td> 2-propanol</td>
 <td>Sigma-Aldrich</td>
 <td>34965-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> Methanol 215 SpS </td>
 <td>Romil</td>
 <td> H409-2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>02860-2.5L</td>
 <td></td>
 </tr>
 <tr>
 <td> Chloroform SpS</td>
 <td>Romil</td>
 <td>H135 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> n-Hexane 95% SpS</td>
 <td>Romil</td>
 <td> H389 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Acetonitrile 230 SpS</td>
 <td>Romil</td>
 <td>H047 2,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Dichloromethane SpS</td>
 <td>Romil</td>
 <td>H2022,5 L</td>
 <td></td>
 </tr>
 <tr>
 <td> Carbon tetrachloride</td>
 <td>Sigma-Aldrich</td>
 <td>107344-1L </td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>383112-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium hydrogen carbonate</td>
 <td>Carlo Erba</td>
 <td>478536-500 g </td>
 <td></td>
 </tr>
 <tr>
 <td> Diethyl ether</td>
 <td>Sigma-Aldrich</td>
 <td> 309966-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S7653-5KG</td>
 <td></td>
 </tr>
 <tr>
 <td> NaOH solid</td>
 <td>Sigma-Aldrich</td>
 <td>221465-25G</td>
 <td></td>
 </tr>
 <tr>
 <td> NH4OH sol. 28%-30%</td>
 <td>Sigma-Aldrich</td>
 <td>221228-1L-A</td>
 <td></td>
 </tr>
 <tr>
 <td> Acetic acid</td>
 <td>Sigma-Aldrich</td>
 <td>320099-500ML</td>
 <td></td>
 </tr>
 <tr>
 <td> Ammonium Cerium(IV)sulfate dihydride</td>
 <td>Sigma-Aldrich</td>
 <td>221759-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Ammonium Molybdate tetrahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>A7302-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sulfuric Acid 95%-98%</td>
 <td>Sigma-Aldrich</td>
 <td>320501-1L</td>
 <td></td>
 </tr>
 <tr>
 <td> Silver Nitrate</td>
 <td>Sigma-Aldrich</td>
 <td>209139-25G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium sulfate anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>238597-500G</td>
 <td></td>
 </tr>
 <tr>
 <td> Nuclease P-I from penicillium citrinum</td>
 <td>Sigma-Aldrich</td>
 <td>N8630-1VL</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphodiesterase II type I-sa </td>
 <td>Sigma-Aldrich</td>
 <td>P9041-10UN</td>
 <td></td>
 </tr>
 <tr>
 <td> Erythro-9-(2-hydroxy-3-nonyl)adenine, hc</td>
 <td>Sigma-Aldrich</td>
 <td>E114-25MG</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphatase alkaline type VII-t from*bov</td>
 <td>Sigma-Aldrich</td>
 <td>P6774-1KU</td>
 <td></td>
 </tr>
 <tr>
 <td> Phosphodiesterase I type VI</td>
 <td>Sigma-Aldrich</td>
 <td>P3134-100MG</td>
 <td></td>
 </tr>
 <tr>
 <td> Deoxyribonuclease II type IV from*porcin</td>
 <td>Sigma-Aldrich</td>
 <td>D4138-20KU</td>
 <td></td>
 </tr>
 <tr>
 <td> Trizma(r) base, biotechnology performanc ce</td>
 <td>Sigma-Aldrich</td>
 <td>T6066-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E1644-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Succinic acid bioxtra</td>
 <td>Sigma-Aldrich</td>
 <td>S3674-250G</td>
 <td></td>
 </tr>
 <tr>
 <td> Calcium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>C5670-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Formic acid, 98 %</td>
 <td>Sigma-Aldrich</td>
 <td>06440-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td> Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane</td>
 <td>Millipore</td>
 <td>UFC500324</td>
 <td></td>
 </tr>
 <tr>
 <td> 8-Bromo-2'-deoxyguanosine</td>
 <td>Berry &amp; Associates</td>
 <td>PR3290-1 g</td>
 <td></td>
 </tr>
 <tr>
 <td> 8-Bromo-2'-deoxyadenosine</td>
 <td>Berry &amp; Associates</td>
 <td>PR3300-1 g</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>383112-100G</td>
 <td></td>
 </tr>
 <tr>
 <td> Sodium hydrogen carbonate</td>
 <td>Carlo Erba</td>
 <td>478536-500 g </td>
 <td></td>
 </tr>
 <tr>
 <td> 2'-deoxyguanosine:H2O (U-15N5, 96-98%)</td>
 <td>Cambridge Isotope Laboratories, Inc</td>
 <td>CILNLM-3899-CA-0.1</td>
 <td></td>
 </tr>
 <tr>
 <td> 2'-deoxyadenosine (U-15N5, 98%) 95%+ CHEMICAL PURITY</td>
 <td>Cambridge Isotope Laboratories, Inc</td>
 <td>CILNLM-3895-0.1</td>
 <td></td>
 </tr>
 <tr>
 <td> Nitrous oxide (N2O) </td>
 <td>Air Liquide </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td> Deoxyribonucleic acid from calf thymus</td>
 <td>Sigma Aldrich</td>
 <td>D4522-5MG</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>60Co-Gammacell</td>
 <td>AECL- Canada </td>
 <td>220</td>
 <td></td>
 </tr>
 <tr>
 <td>Immersion well reaction medium pressure 125 watts </td>
 <td>Photochemical reactors ltd</td>
 <td>Model 3010</td>
 <td></td>
 </tr>
 <tr>
 <td>Evaporating flask 250 ml</td>
 <td>Heidolph</td>
 <td>P/N NS 29/32 514-72000-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Luna 5 &mu;m C18(2) 100 &Aring;, LC Column 250 x 4.6 mm </td>
 <td>Phenomenex</td>
 <td>00A-4252-E0 </td>
 <td></td>
 </tr>
 <tr>
 <td>Alltima C8 Column 250 x 10 mm 5 &mu;m</td>
 <td>Grace</td>
 <td>88081</td>
 <td>Semipreparative</td>
 </tr>
 <tr>
 <td>SecurityGuard Kit</td>
 <td>Phenomenex</td>
 <td>KJ0-4282</td>
 <td>Analytical holder kit and accessories</td>
 </tr>
 <tr>
 <td>Holder for 10.0 mm ID cartridges</td>
 <td>Phenomenex</td>
 <td>AJ0-7220</td>
 <td>Semipreparative holder </td>
 </tr>
 <tr>
 <td>10.0 mm ID cartridges </td>
 <td>Phenomenex</td>
 <td>AJ0-7221 </td>
 <td></td>
 </tr>
 <tr>
 <td>High-performance liquid chromatography (HPLC) </td>
 <td>Agilent</td>
 <td>1100</td>
 <td></td>
 </tr>
 <tr>
 <td>LC/MS/MS </td>
 <td>Applied Biosystems</td>
 <td>4000QTRAP System</td>
 <td></td>
 </tr>
 <tr>
 <td>Tandem mass ESI spectrometer </td>
 <td>(Bruker Daltonics) </td>
 <td>Esquire 3000 plus </td>
 <td></td>
 </tr>
 <tr>
 <td>Vial 2-4 ml </td>
 <td>SUPELCO </td>
 <td>Cod 27516</td>
 <td></td>
 </tr>
 <tr>
 <td>Vial 4 ml </td>
 <td>SUPELCO </td>
 <td>Cod 27517</td>
 <td></td>
 </tr>
</tbody>
</table>

free radicals in chemical biology: from chemical behavior to biomarker development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

The isomerization process has been described in particular for cholesteryl esters affording the mono-trans isomers of linoleic and arachidonic acids shown in Figure 1 as the first products of this attack, which can occur under conditions of free radical stress in the biological environment.5
In Figure 3 the chemical mechanism involved in the cis-trans double bond isomerization is shown. The radical source is indicated in a generic way to afford S-centered radicals. In the described protocols the radical source is UV light that is able to break homolitically the S-H bond present in the thiol molecule RSH. 
Figure 4 summarizes the three steps protocol for the synthesis of the modified lipid class and their detection in human plasma: the synthesis represents a biomimetic free radical process and also provides a one-pot convenient entry to the geometrical isomers, without any contamination by positional isomers followed by purification and isolation protocols.
Several analytical methodologies can be applied for a high sensitive detection of the trans isomer content and characterization of the mono-trans cholesteryl ester library. In particular, Raman spectroscopy can be carried out directly on the cholesteryl ester fraction without derivatization (see Figure 2 and Figure 9). 
The second example concerns purine 5',8-cyclo-2'-deoxyribonucleosides, which are DNA lesions created by the attack of free radicals to the position C5' of the sugar moiety and subsequent formation of a covalent bond between the sugar and the base moieties. Four structures can be produced, that is, 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine, both existing in the 5'R and 5'S diastereomeric forms (Figure 5). In Figure 6 the reaction mechanism is shown, that involves photolysis of 8-bromopurine derivatives 5 to give the corresponding C8 radical 6, which intramolecularly abstracts a hydrogen atom from the C5' position selectively affording the 2'-deoxyadenosin-5'-yl radical 7. Radical 7 undergoes cyclization with a rate constant in the range 105-106 s-1,2 followed by oxidation of the heteroaromatic radical 8 to give the final products 9. The reaction of hydroxyl radicals, generated by radiolysis of water, with 2&#x2032;-deoxyadenosine and 2&#x2032;-deoxyguanosine (10) was found to occur ca. 10% by hydrogen abstraction from C5&#x2032; position.7 
Our chemical biology approach for the libraries of purine 5',8-cyclo-2'-deoxyribonucleosides (including labeled compounds) is illustrated in Figure 7, with the identification of these lesions in oligonucleotides as well as in DNA samples obtained from various sources such as, for example, treated under ionizing radiation conditions, as mimic of radical stress conditions.
In Figure 8 a typical photoreactor equipment is shown. The device allows for irradiation of compounds dissolved in the appropriate solvent. It consists of: i) the reaction chamber equipped with the inlet for the inert gas (on the bottom) and two inlets for the gas exit and for the reagents addition; ii) an internal chamber containing the appropriate mercury lamp connected to a cooling system and electrical power, which is inserted into the reaction chamber through a glass joint. 
<img alt="Figure 1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50379/50379fig1highres.jpg" src="/files/ftp_upload/50379/50379fig1.jpg" /> 
Figure 1. Mono-trans isomers of cholesteryl linoleate and arachidonate.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50379/50379fig2highres.jpg" src="/files/ftp_upload/50379/50379fig2.jpg" /> 
Figure 2. Cholesteryl esters in human serum and direct analysis for mono-trans isomers by Raman spectroscopy.
<img alt="Figure 3" fo:content-width="4in" fo:src="/files/ftp_upload/50379/50379fig3highres.jpg" src="/files/ftp_upload/50379/50379fig3.jpg" /> 
Figure 3. The addition-elimination process that leads to the cis-trans isomerization of double bonds by thiyl radicals.
<img src="/files/ftp_upload/50379/50379fig4.jpg" alt="Figure 4" /> 
Figure 4. Three steps protocol for development of mono-trans cholesteryl esters as biomarker of free radical stress.
<img alt="Figure 5" fo:content-width="6.5in" fo:src="/files/ftp_upload/50379/50379fig5highres.jpg" src="/files/ftp_upload/50379/50379fig5.jpg" /> 
Figure 5. The four purine 5',8-cyclo-2'-deoxyribonucleoside diastereoisomers. <a href="https://www.jove.com/files/ftp_upload/50379/50379fig5large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 6" fo:content-width="6.5in" fo:src="/files/ftp_upload/50379/50379fig6highres.jpg" src="/files/ftp_upload/50379/50379fig6.jpg" /> 
Figure 6. Generation of C5' radicals, either by photolysis of 5 or by reaction of 10 with HO&bull; radicals, and the mechanism of purine 5',8-cyclo-2' deoxyribonucleoside formation. <a href="https://www.jove.com/files/ftp_upload/50379/50379fig6large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50379/50379fig7.jpg" alt="Figure 7" /> 
Figure 7. Protocol for identification of some radical-based DNA lesions; mtDNA: mitochondrial DNA, nDNA: nuclear DNA.
<img src="/files/ftp_upload/50379/50379fig8.jpg" alt="Figure 8" /> 
Figure 8. Photochemical reactor.
<img src="/files/ftp_upload/50379/50379fig9.jpg" alt="Figure 9" /> 
Figure 9. Representative Raman spectrum of plasma cholesteryl ester. In the inset the comparison of a specific region with cholesteryl linoleate and cholesteryl mono-trans linoleic acid isomers.
<img src="/files/ftp_upload/50379/50379fig10.jpg" alt="Figure 10" /> 
Figure 10. Representative GC analysis of FAME obtained from plasma cholesteryl esters.
<img alt="Figure 11" fo:content-width="4.5in" fo:src="/files/ftp_upload/50379/50379fig11highres.jpg" src="/files/ftp_upload/50379/50379fig11.jpg" /> 
Figure 11. Representative HPLC run containing 2'-deoxyadenosine and 2'deoxyguanosine together with their oxidative and purine 5',8-cyclo-2'-deoxyribo nucleosides. <a href="https://www.jove.com/files/ftp_upload/50379/50379fig11large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development at JoVE.com

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

A protocol for the synthesis, purification, and characterization of a ruthenium-based inhibitor of mitochondrial calcium uptake is presented. A procedure to evaluate its efficacy in permeabilized mammalian cells is demonstrated.

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.