Synthesis of New Antioxidant Phenolic Acid Derivatives
By Abdelkader MEBARKI Tutor: Jorge GARRIDO Training course place: CIETI (ISEP Portugal)
April-juny 2010
Anknowledgements: At first I really want to express my gratitude to Mrs. Ana Paula Tavarez for accepting me in ISEP Company. Big thanks to my tutor M. Jorge Guarrido for having accompanied me in all the training session during. Special thanks to Manuela Guarrido, all master students who sheared the laboratory with me for their kindness and cheerfulness. Finally, I thank all the ISEP employees.
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Summary: Presentation of ISEP……………………………………p3 Preface……………………………………………………p4 I] Introduction: theory……………………………………….p5 to p8 1) Lipid oxidation…………………………………….........….p5 to p6 2) Antioxidant………………………………………………….p6 to p7 a) First case: preventive antioxidants…………………………………………p6 b) The second case: chain breaking antioxidants……………..………p6 to p7
3) The phenolic compounds………………………………………p7 4) Synthesized compounds during the course…………………...p8 a) Synthesized compound 1……………………………………………………p8 b) Synthesized compound 2……………………………………………………p8
II] Recalls………………………………………………...p9 to p13
1) TLC……………………………………………………….p9 to p11 2) Nuclear Magnetic Resonance…………………………………………p11 to 13 a) Proton NMR Spectroscopy………………...………………………p11 to p12 b) Carbon NMR Spectroscopy…………………………………….....p12 to p13
III] lab work………………………………………………p13 to p24 1) choice of the eluent…………………………………………….p13 2) synthesize of first compound…………...………………p14 to 24 a) first try………………………………………………………………….p14 to 20 b) second try……………………………………………………………..p20 to 24
Conclusion…….………………………………………………….p25 Annex 1: proton nmr spectroscopy Annex 2: carbon nmr spectroscopy Annex 3: 5-(3-Chloro-4,5-dimethoxyphenyl)-2,4-pentadienoic Acid procedure Annex 4: table of product used during the course
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Presentation of ISEP
The Instituto Superior de Engenharia do Porto (ISEP) is part of Instituto Politécnico do Porto (IPP). The IPP built in 1985, and recoveries the polytechnic education which began in 1979. Currently IPP is composed by 7 schools
Escola Superior de Educação (ESE) Escola de Música (ESM) Instituto Superior de Engenharia (ISEP) Instituto Superior de Contabilidade e Administração (ISCAP) Esclola Superior d’Estudos Industriais e de Gestão (ESEIG) Escola Superior de Tecnologias e Gestão de Felgueiras (ESRGF) Escola Superior de Tecnologias da Saúde (ESTSP)
ISEP have built in 1852, during the rise of Portuguese liberalism. It belong to IPP since 1988. There are around 5600 students and, 9 departments. The chemistry department has started in 1974. In chemistry department there are 8 research groups. I have work in CIETI ( Centro de Inovação em Engenharia e Tecnologia Industrial ) promotes research in order to create, develop and characterise new products, processes and systems which will contribute to the innovation in industry.The group supports scientific education in Instituto Superior de Engenharia do Porto integrating in its projects MSC and PhD students. CIETI is multidisciplinary research group of the Instituto Superior de Engenharia do Porto. Its research activities are divided in four fields:
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Biomaterials and Nanotechnolgies
Environment and Energy
Production Technologies, Chemical Processes and Biotechnology
Process Control, Information and Communication Technologi
Preface: Free radicals are chemical species with an unpaired electron. These species are harmful if present in higher amounts, leads to quality deterioration of foods and cosmetics and could have harmful effects on health (Alzheimer’s disease, cancers, inflammation, aging, etc….) because they are involvement in lipid oxidation especially that induced by reactive oxygen species (ROS). The solution against this phenomenon is the use of antioxidants, substances able to deactivate free radicals. There are natural and synthetic antioxidants. The natural antioxidant are mainly present in cereals, fruits, vegetables, beverages and in the human organism like gluthation, bilirubin, estrogenic sex hormones, uric acid, coenzyme Q, melanin, melatonin, α-tocopherol and lipoic acid… However the natural antioxidants sometimes are not sufficient, particularly when conditions like heat and, light involve oxidative stress phenomenon, caused by an imbalance between antioxidant systems and the production of oxidants. So, nowadays the quest of new antioxidants is an important area of research in the field of Biological and Biochemical sciences. As a consequence of the research group current findings on the structure-property-activity of phenolic acid antioxidants, in this project new antioxidant phenolic acid derivatives will be synthesized with a new structure that could be produce more efficient antioxidant than the already existing.
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I] Introduction: theory I] 1) Lipid oxidation Oxidation is A reaction in which the atoms in an element lose electrons and the valence of the element is correspondingly increased. Lipid oxidation is induced in the presence of free radicals initiators such as heat, light, photosensitizing pigments and metal ions. In this condition triplet Oxygen, 3O2, which could be considered as a ground-state biradical •OO•, and singlet Oxygen, 1O2, which corresponds to an excited state of the molecule, are formed. The singlet oxygen formed is electrophilic and can thus bind directly to fatty acids double bond, leading to hydroperoxide formation. Lipid oxidation reaction is composed of three phases (initiation, propagation and termination). Initiation: L1H → L1• + H• Initiation results in the formation of L1• free radicals, these are highly unstable, shortlived intermediates that stabilize by abstracting a hydrogen from another chemical species.
Propagation: Radicals formed during the initiation phase reacts very quickly with oxygen to generate different radical species, hydroxyl (HO•) and hydroperoxyl (HOO•) radicals, alkoxyl (LO•) and peroxyl (LOO•) radicals. L1• + 3O2 → L1OO• L1OO• + L2H → L1OOH + L2• This so-called “self-sustained” radical chain reaction thus propagates at a high rate. Many hydroperoxide isomers are thus formed during this phase. So many fatty acids molecules are oxidized. Hydroperoxide formation from unsaturated fatty acids is generally accompanied by stabilization of the radical state via double-bond
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rearrangement (electron delocalization), which gives rise to conjugated dienes and trienes. Termination: The oxidation process then continues with the transformation of hydroperoxides into secondary nonradical oxidation compounds. The main hydroperoxide decomposition mechanism involves scission of the double-bond adjacent to the hydroperoxyl group, leading to the formation of hydrocarbons, aldehydes, alcohols and volatile ketones. The reaction can then also terminate after polymer formation. Moreover, many antioxidants can facilitate termination of radical chain oxidation.
I] 2) Antioxidant These have two different ways to counteract oxidation: by protecting target lipids from oxidation initiators or by stalling the propagation phase. In the first case, the so-called preventive antioxidants hinder ROS formation or scavenge species responsible for oxidation initiation (O2•-, 1O2, etc.). In the second case, the so-called ‘chain breaking’ antioxidants intercept radical oxidation propagators (LOO•) or indirectly participate in stopping radical chain propagation.
I] 2) a) Preventive antioxidants There are many different “preventive” antioxidation pathways because of the diverse range of available oxidation initiators. These pathways include chelation of transition metals, singlet oxygen deactivation, enzymatic ROS detoxification, UV filtration, inhibition of proxidant enzymes, antioxidant enzyme cofactors, etc. Here, we will only singlet oxygen deactivation will be described because is one of He most important.
Nowadays carotenoids are the most efficient molecules for 1O2 quenching. In addition to carotenoids, there are other 1O2 quenchers, these are phenolic acid derivatives especially tocopherols and thiols. This quenching mechanism of action occurs through deactivation of 1O2 into 3O2. Many antioxidants like phenolic acid derivatives, have antioxidant activity through several different but highly complementary mechanisms, i.e. chain breaking antioxidants and 1O2 quenchers. However the most of phenolic acid derivatives are chain-breaking antioxidants.
I] 2) b) Chain-breaking antioxidants In lipid peroxidation, chain-breaking antioxidants usually lose a hydrogen radical to LOO• thus halting radical oxidation propagation.
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Chain-breaking mechanism: A-H + L•
→ A• + LH
A-H + LOO• → A• + LOO-H A-H + LO•
→
A• + LOH
A• + LOO• → LOOA A• + LO• → LOA This primarily involves mono- or poly-hydroxylated phenol compounds (tocopherols, tocotrienols, flavonoids, phenolic acids and alcohols, stilbenes, etc.) with different substituents on one or several aromatic rings. This way is under multifactorial control: -
the capacity of a phenol to dispose of an H atom could be quantified by the homolytic dissociation energy of the O–H bond, i.e. bond dissociation energy (BDE). More weak is BDE more efficient is the phenolic compound.
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A suitable position and good mobility towards LOO• production sites are key attributes which ensure that an antioxidant will be a good chain breaking antioxidant.
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Finally, the reactivity of antioxidant derived radicals with unsaturated lipids should also be taken into account. This fate is generally dictated by the capacity of the antioxidant to stabilize unpaired electrons by delocalization.
I] 3) The phenolic compounds OH R
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R R
1
R R
3
2
Along with the antioxidant activity, phenols are also used as chemopreventive or antiinflammatory agents. As antioxidant compound their consumption has become essential in our diet. These are present in the plant (red berries, garlic, tea, grappes etc…).
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The majority of phenols are chain-breaking antioxidants thanks to the presence of electron donor group like OH on the aromatic ring. Studies have shown that phenolic antioxidants activity depends on the geometry of the molecule and, its structure, i.e. the number of constituent and the type of substituents.
I] 4) Synthesized compounds during the course I] 4) a) Synthesized compound 1:
So we can see on the Antioxidant phenolic derivative 1 two conjuged double bounds, And two electron donor groups “OH”. So it will be able to stabilize unpaired electrons by OH presence and delocalization.
I] 4) b) Synthesized compound 2:
This second compound has three electron donor group “OH”. Theoretically the second antioxidant will be more efficient.
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The procedure used in this project to synthesize the phenolic acid derivatives, some organic chemistry procedures and techniques were used, TLC, column chromatography and NMR, that are recalled.
II] Recalls II] 1) TLC
TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferably both run on the same TLC plate). A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate.) The procedure for TLC, explained in words and is illustrated with photographs in the next First of all prepare the eluent, 10 ml of eluent were measured in a graduated cylinder The prepared chamber was left to saturate during 10 minutes.
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Put one or two drop with capillar in the middle of the cross, then let dry. If the sample to analyse is, dissolve it in a small amount of organic solvent.
Insert the plate in the container and wait until elution is complete, seen line like you see in the above picture.
TLC plate was left to dry and the spots were seen by using a UV lamp (254nm). And see if there are Two spots which have the same retention factor Rf= h/H see the example in the next page.
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The TLC is a test very easy. We see in this TLC plate that the compound number one and number two are different because their retention factor aren’t same but these two compounds are pure because there is only one spot for each . However the compounds number 3 is a mixture of compound number one and two.
II] 2) Nuclear Magnetic Resonance Over the past fifty years nuclear magnetic resonance spectroscopy, commonly referred to as nmr, has become the preeminent technique for determining the structure of organic compounds. Of all the spectroscopic methods, it is the only one for which a complete analysis and interpretation of the entire spectrum is normally expected. Although larger amounts of sample are needed than for mass spectroscopy, nmr is non-destructive, and with modern instruments good data may be obtained from samples weighing less than a milligram. Here we see Proton NMR Spectroscopy and Carbon NMR Spectroscopy. In these nmr tetramethylsilane, (CH3)4Si, usually referred to as TMS has become the reference.
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II] 2) a) Proton NMR Spectroscopy Each proton gives a particularly signal. Its signal depends of number of protons, the spin-spin interactions and the atom which bound with these protons (cf annex 1). So about spin-spin interactions: This is a common feature in the spectra of compounds having different sets of hydrogen atoms bonded to adjacent carbon atoms. The signal splitting in proton spectra is usually small, ranging from fractions of a Hz to as much as 18 Hz, and is designated as J (referred to as the coupling constant). The splitting patterns found in various spectra are easily recognized, provided the chemical shifts of the different sets of hydrogen that generate the signals differ by two or more ppm. The patterns are symmetrically distributed on both sides of the proton chemical shift, and the central lines are always stronger than the outer lines. The most commonly observed patterns have been given descriptive names, such as doublet (two equal intensity signals), triplet (three signals with an intensity ratio of 1:2:1) and quartet (a set of four signals with intensities of 1:3:3:1). Four such patterns are displayed in the following illustration. The line separation is always constant within a given multiplet, and is called the coupling constant (J). The magnitude of J, usually given in units of Hz, is magnetic field independent (cf annex 1).
II] 2) b) Carbon NMR Spectroscopy: The power and usefulness of 1H nmr spectroscopy as a tool for structural analysis should be evident from the past discussion. Unfortunately, when significant portions of a molecule lack C-H bonds, no information is forthcoming. That’s why there is Carbon nmr spectroscopy. Unlike proton nmr spectroscopy, the relative strength of carbon nmr signals are not normally proportional to the number of atoms generating each one. Because of this, the number of discrete signals and their chemical shifts are the most important pieces of evidence delivered by a carbon spectrum. The general distribution of carbon chemical shifts associated with different functional groups (cf annex 2).
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So after these theorie, these recalls we can talk about my lab work. That is to say we see in the next part the procedure to synthesize the products, and the difficulties that we met.
III] lab work The procedure used to synthesize the new antioxidant compounds was adapted from the literature and is included in annex 3.
III] 1) choice of the eluent: Three different eluents were tested in order to follow the reaction.
As can been the left’s TLC is not appropriate because the retention factor of compound is too low. So we can use CHCl3/MeOH (methanol) 9:1 or AcoEt (ethylacetate) / DCM (dicloromethan) 8:2. We have use CHCl3/MeOH 9:1.For the column chromatography, we have use DCM/MeOH
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III] 2) synthesize of first compound III] 2) a) first try:
Round flask of 100 ml containing:
1 g of 3,4‐ dimethoxibenzaldehyde 0.67 g of potassium tert‐ butoxide 0.83 ml of ethylcrotonate +0.20ml after 2h of stir 6.5ml of pyrrolidinone
We followed this reaction by TLC. The reaction was stopped when the TLC showed that the aldehyde used as reactant was disappeared:
As we see in the TLC the aldhyde has disappeared in the following day.
We acidified with concentrate HCL with Pasteur pipette in little bit of ice:
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Then we can begin the extraction:
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We acidified carefully the organic phase 2 with concentrate HCL with a Pasteur pipette.
We filter this solid (S1): We do a TLC. We can see the product is impure.
After this filtration we have wanted to see if there were solid in organic phase, so we have done an extraction a second time with NaHCO3. After that we got a new phase aqueous. And we have done the same procedure with this phase: acidification and filtration. We have done a TLC of this solid (S2).
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As you see on the TLC the S2 solid that we have got after the second time extraction with NAHCO3,is pure
Crystallization of solid S2:
We have done a TLC test but there were no spot. And we have tried to do an nmr but that didn’t work, this solid didn’t dissolve in all organic solvent. We have concluded that this solid is the potassium tert-butoxide. So we have failed, in the next part we see the new procedure.
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After this failure we have tried to purify the solid S1with a column chromatography.
In a beaker put some silica‐gel then add small amount of CH2Cl2 at the end put the beaker’s contain in the column. Put in the beaker the solid S1 with small amount of silica‐gel and CH2Cl2.
So after that we have been able to collect the fractions first in the round flask and after evaporation you can put in a tube. In order to evaporate the solvent you program the pump for the solvent that you want to evaporate and use the Büchi rotavapor here CH2Cl2 (60°C, atmospheric pressure). See the photo above:
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And we have done TLC of fractions collected. After this TLC we have been able to conclude that the fractions
1 to 3 it was just CH2Cl2 same for 7 to 14. 4 to 6 contained aldehyde 15 and 16 have been join because these contained the same impure product
17 to 20 was just CH2Cl2 21 to 31it was an unknown pure product, we have joined these fractions.
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So we have put 21 to 31 on ependorf and we have analyzed it and we notice that the product obtained was impure.
III] 2) b) second try: This second try it’s a new procedure without extraction.
Round flask of 100 ml containing:
5 g of 3,4‐ dimethoxibenzaldehyde 3.35 g of potassium tert‐ butoxide 4.15 ml of ethylcrotonate 32.5ml of pyrrolidinone
We have begun this reaction at the beginning of the day and at the end we have put n the fridge until the following day, amazing this following we have seen in there deep of ball flask solid was formed. After this discovery we have had to adapt, look at the procedure above
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Then we have begun a colon chromatography. And we have done TLC of fractions collected. After this TLC we have been able to conclude that the fractions
1 to 5 it was just CH2Cl2. 5 to 50 contained aldehyde 51 to 54 was just CH2Cl2 55 and 56 have joined because these contained the same impure product
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57 to 90 it was an unknown pure product, we have joined these fractions. Without acetic acid this spot is so spread
With some drop of acid acetic in TLC ‘s eluent. You can see this product is not totally pure.
After that we have joined 57 to 90 we have evaporated the solvent of fraction. We have got a solid. Then we have washed it. Washing (do this manipulation three times):
We have dried it with a deschicator. Look this beautiful aspect above (it’s shining, in this try we have got crystal)
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NMR analyzes:
Zoom in aromatics region:
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Zoom in aromatics region:
So these analyzes confirm that this solid is well our compound and the purities of product.
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Conclusion We have success to synthesize the middle compound of first antioxidant that we have wanted. To get this compound is indispensable to use colon chromatography, this procedure is very long, take one week. Probably this procedure work to make the middle compound of the second antioxidant. It is shame that the time missed us for the column to make the second synthesized. I hope that someone go on my work with success. I have learnt with this training course theoretical and practical knowledge in chemistry. Theoretically I have learnt pointed knowledge about antioxidant their mechanism, the groups which give to molecule an antioxidant activity…and I have learnt some knowledge about the carbon nmr spectroscopy. Practically I have learnt to do a colon chromatography, to use the rotatif evaporator. I have improved about TLC. I have seen the nmr spectroscopy in university of Porto. And I have improved my English. Moreover I have visited Porto I have improved my self-government. This training course was totally conform with my chemistry knowledge
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