The overall goal of this experiment is to provide models of the free radical transformation of biomolecules. The chemical, mechanistic and analytical information can then be transferred to biology, medicine and biomarker discovery. This is achieved by first synthesizing appropriately modified biomolecules under free radical conditions initiated by UV light.
The synthetic plan can also be designed to simulate free radical processes occurring in a biological environment, which is the principle of biomedical radical chemistry. As a second step, the products are purified, analyzed and characterized, which provides structural and chemical information of the modified biomolecules in order to organize molecular libraries and facilitate recognition in the complexity of a biological environment. The protocols used enable the separation of complex mixtures of compounds such as those derived from biological specimens.
Finally, in vitro cell culture experiments and in vivo studies from animals and human are used to develop biomarkers. These results show the importance of chemical biology studies of free radical reactions to the understanding of complex transformations, which can be the basis of signaling and damages in living organisms. Our group has an an interdisciplinary approach leaking chemistry and chemical skills to biological processes towards health application.
We first study chemical mechanism using appropriately designing biome medic scenarios for which we development a library of modification of biologically related molecules due to the free radical process. Then we s shift to more complex studies in biological samples ing the presence of modify compounds as an effect of free radical attack. Often free radical stress in living organisms lacks reliable and feasible chemical mechanisms to explain their effects.
This approach allows the exchange of chemical knowledge with other life sciences disciplines creating a common ground for the full understanding of biological processes. This is a very important training for the new generation of scientists that must have a multi and interdisciplinary approach to face the research challenges of molecular sciences in the 21st century. Demonstrating the procedures will be the five researchers working in our lab.
Anna, permanent researchers in our institute, Michel Postdoc with the grant from Italian Ministry of Education, Mika to Mauri fellows and postdoc in our group arm to and Annaa Azi, again permanent in our group. Begin by Sonic a 15 Millimolar CHO sterile linoleate Ester propanol solution for 15 minutes. After transferring the solution to a quartz photochemical reactor at 7.6 microliters of a two molar solution of tumor capto ethanol into propanol to it.
Insert the lamp in the reactor and regulate the argonne flux. Flush the reaction mixture with Argonne for 20 minutes. In order to eliminate the presence of oxygen in the solution, irradiate the reaction mixture by UV light using a 5.5 low pressure mercury lamp at 22 plus or minus two degrees Celsius for four minutes after turning off the UV light.
Monitor by analytical silver TLC to detect the formation of the mono trans cho sterile esters. Dip the TLC plate in a cerium ammonium MA date solution and heat until spots appear on the plate. Next, collect the reaction mixture in a round bottom flask.
Wash the reactor with a few milliliters of two propanol and transfer the solution to the same flask. Remove the solvent by rotary of aberration. Purify the mono trans isomers of co steryl linoleate ester by silver TLC as described in the literature.
Use a nine to one volume ratio of hexane dathyl ether as the elu following purification. Dilute one milliliter of human serum with one milliliter of brine and transfer the solution into a separatory funnel under a stream of Argonne. In order to avoid oxidation adducts, add 10 milliliters of chloroform methanol to the funnel.
Shake the separatory funnel very slowly in order to limit emulsion formation. Due to the presence of albumin, allow the two layers to separate and then remove the bottom organic layer from the funnel. Repeat the extraction three times.
Transfer the combined organic layers to the funnel and wash them once with 10 milliliters of brine. Collect the organic layers in an erlenmeyer flask under an argon stream and add anhydrous sodium sulfate. Filter the solution through a glass funnel fitted with cotton.
Transfer the filtrate to a round bottom flask and remove the volatiles by rotary evaporation. Dissolve the crude oil in one milliliter of a two to one volume ratio of chloroform methanol, and load the solution onto a preparative TLC plate under a stream of argonne. Use a mixture of hexane ethyl ether as the eent scrape off the silica portion containing the cholester ester fraction and transfer it to a vial.
Next, extract the silica three times with five milliliters of two to one chloroform methanol. Filter the silica off through a buchner funnel under vacuum and transfer the organic layer to a round bottom. Flask then evaporate the solvent to afford the pure cho sterile esters.
Dissolve the esters in the flask with hexane. Transfer the solution to a vial and evaporate the solvent under an argon stream. Store the choral esters in a dark vial covered by aluminum foil under argon at minus 20 degrees Celsius.
The next step is to dissolve 33 milligrams of eight bromo, two diox adenosine in 100 milliliters of acetonide trial. Regulate the Argonne gas flow at the bottom of the photo reactor and add the solution in it. Then pass a needle through the center of a septum and place it on one of the reactors exits.
Then seal the other photo reacts inlet with another septum flush with inert gas for 30 minutes. Next, adjust the water circulation and turn on the UV light after irradiating with UV light. Using a 125 watt medium pressure mercury lamp at 22 plus or minus two degrees Celsius for 15 minutes.
Collect the solution into a 250 milliliter round bottomed flask. Wash the photo reactor with 10 milliliters of acetonitrile and transfer the washings to the same flask. Next, quench the crude reaction mixture with one molar ammonium hydroxide and evaporate the solvent.
Using a rotary evaporator, run the high performance liquid chromatographic analysis with a C 18 analytical column and compare the profile with reference compounds. Use two millimolar ammonium formate as solvent A and ACEDONITE trial as solvent B.Collect the chromatographic peaks corresponding to the two DIA stereo meric products in two different files. See the text protocol for chromatographic conditions.
Measure the absorbance of the two collected fractions in the UV spectrophotometer and calculate the exact concentration based on the Lambert beer law. Use the extinction coefficient of two prime de adenosine dissolve 35 milligrams of eight, promo two deoxy guine in 100 milliliters of distilled water. Regulate the argon flow and fill the photo.
React with the reaction solution after degassing the reaction mixture as shown earlier. Irradiate by UV light using a 125 watt medium pressure mercury lamp at 22 plus or minus two degrees Celsius for 30 minutes, and collect the solution into a 250 milliliter round bottomed flask. Wash the photo, react with 10 milliliters of water and transfer the washings to the same flask, quench the crude reaction mixture with one molar ammonium hydroxide solution and evaporate the solvent hunter vacuum.
Perform analysis as described earlier. Prepare one milliliter of a 0.5 milligram per milliliter solution of calf thys DNA in distilled water and transfer it to a two milliliter glass vial with a septum cap. Connect a one inch needle to a nitrous oxide gas line, passing it through the center of the septum until it reaches the bottom of the vial.
Then insert a short needle into the septum to surface the gas exit. Flush the solution with nitrous oxide for 30 minutes after degassing. Pull out the short exit needle first followed by the one inch needle so that the pressure remains inside the vial.
Transfer the solution to the gamma radiologist apparatus and irradiate for 30 minutes to an upend DPH tube containing 80 micrograms of DNA. Add a pre-made enzymatic digestion mixture mixed gently by pipetting the mixture up and down. Incubate the mixture at 37 degrees Celsius for 48 hours.
Next, add a pre-made mixture of alkaline phosphatase and phosphodiesterase one interest HCL buffer to the digestion mixture mixed gently by pipetting the mixture up and down. Incubate the mixture at 37 degrees Celsius for two hours. Neutralize the mixture by adding five microliters of 10%formic acid to a centrifugal filter fitted in a micro centrifuge tube.
Add the sample and 200 microliters of tri distilled water centrifuge the contents at 14, 000 cheeses for 15 minutes. Then separate the filter device from the micro centrifuge tube. Put parfum on the micro centrifuge tube and freeze it in liquid nitrogen.
Then lyophilize the enzyme free solution in the micro centrifuge tube. The chemical mechanism involved in the cyst trans double bond isomerization is shown here. The radical source is indicated in a generic way to afford S centered radicals.
In the described protocol, UV light serves as the initiator and holi breaks the SH bond in tumor capto ethanol. The three step protocol for the development of the modified lipid class consists of the synthetic strategy, the purification and characterization of these compounds and their detection in human plasma. The synthesis represents a biomimetic free radical process and also provides a one pot convenient entry to geometrical isomers without any contamination by positional isomers.
Several analytical methodologies can be used for high sensitive detection of the trans icier content and characterization of the mono transsterile ester library. In particular, ramen spectroscopy can be carried out directly on the choral ester fraction without derivitization. A representative ramen spectrum of cho esters is shown here.
The inset shows a specific region of the spectrum that compares the Cho steryl linoleate and cho steryl mono trans linoleic acid isomers purine five prime eight cyclo. Two prime diox ribonucleotides are DNA lesions created by the attack of free radicals to the C five position of the sugar moiety and subsequent formation of a covalent bond between the sugar and the base moieties. Four DIA stereos can be formed in this reaction.
Five prime eight cyclo, two prime diox adenosine, and five prime eight cyclo, two prime deoxy guine, both existing in the five prime R and five prime S forms. The reaction mechanism for photo lysis of eight bromo purine derivatives is shown here. Photo lysis of eight bromo purine derivative five affords the corresponding C eight radical six, which then intra molecularly abstracts a hydrogen atom from the C five prime position to selectively yield radical seven.
Radical seven undergoes cyclization followed by oxidation of the hetero aromatic radical eight to give compound nine reaction of compound 10 with hydroxy radicals generated by radiologists of water. Occurred about 10%by hydrogen abstraction from the C five position. The chemical biology approach for the libraries of purine five prime eight cyclo, two prime deoxyribo nucleosides is illustrated here with the use of the molecular library previously described for the identification of these lesions in oligonucleotides, as well as in DNA samples obtained from various sources.
A representative example of lc analytical recognition of DNA nucleosides and their oxidative and cyclo derivatives is shown here. While attempting this procedure, it's important to remember the efficiency of analytical equipment like L-C-M-S-A-G-C must be appropriately checked by calibrations of reference compounds. In addition, satisfactory separation of the compound is important to achieve when a biological sample is analyzed.
After watching this video, you should have a good understanding of how to connect chemical knowledge and experimental skills with biological systems, transferring the identification of molecular libraries or radical based modifications to the biomarker discovery and assessment of cellular stress.
