This report is submitted in part of fulfilment of the requirements for the final year project for the degree of Bsc (Hons) Chemistry at the Manchester Metropolitan University. None of the work in my project described has been presented in support of an application for another degree at this or any other university.
The aim of the project is to synthesise Acridine Orange and derivatives by using existing methods. Fluorescence can be undertaken using light by exposing the sample using UV light.
The compound would be made in the same manner as using the Bernsthen method but using N, N -dimethyl-m-phenylenediamine and 4-chloro-2-benzoic acid to make the diphenylamine with the N-methyl groups on the 3, 6 positions of the rings.
4-chloro-2-benzoic acid and N, N-dimethyl-m-phenylenediaime was reacted together in the presence of tetrohydranfuran which was reacting with N, N diisopropylethylamine. The experiment carried out did not work due to the chlorine being very electronegative pulling the electrons towards it. Also the compound is does not contain enough EWG groups that draw the electrons away from the reaction centre hence the reaction not taking place.
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The purpose of the project is to synthesise Acridine Orange using the current processes that are used to synthesise Acridine and then replace one of the methyl groups on the Nitrogen in Acridine Orange by substitution. This compound is a heterocylic compound containing nitrogen's therefore, a metal compound can be added to that position in the compound and its properties can be obtained and evaluated.
1.1. Intro to acridine
Graebe and Caro discovered Acridine in 1870, in coal tar of high boiling fractions.1 Acridine has a melting point of around 1110C and is cream in colour. This compound has an intense fluorescence of a violet colour.
1.2. Physical properties
Acridine has many properties E.g. fluorescence, ultraviolet spectra, surface activity and association, ionisation, dipole moments.
Fluorescence is the name given to the process when a molecule reaches its electronic excited state by light hitting the molecule.
HÎ½ = photon energy with h = PlanckHYPERLINK "http://en.wikipedia.org/wiki/Planck's_constant"'HYPERLINK "http://en.wikipedia.org/wiki/Planck's_constant"s constant and Î½ = frequency of light.
State S0 is called the ground state of the fluorophore (fluorescent molecule
S1 is its first (electronically) excited state.
Fluorescence is usually found in substances that have a conjugated cyclic ring. These conjugated rings are rigid and when the light is emitted from the molecule, it is longer in wavelength, which is what is stated in Stoke's Law.2
Fd is the frictional force (in N),
Î¼ is the fluid's dynamic viscosity (in Pa s),
R is the radius of the spherical object (in m), and
V is the particle's velocity (in m/s).
Fluorescence does not depend much on the absorbed light with a longer wavelength.3 Much of the fluorescence cannot be seen by the naked eye E.g. naphthalene is invisible due to having light absorbed in the 300mÂµ that lies in the ultraviolet region hence the fluorescence that has been excited not being shown.
A molecule which has not been excited and is in the normal phase contains the 2 lowest orbital to be filled by two opposite spin electrons in the singlet state. When light is exposed on to the molecule, one electron is excited and rises without spin state changing to a higher level.
This excited singlet state is produced and when this excited molecule returns to the ground state, the emission of light given off when returning is known as the fluorescence.
Most acridines show fluorescence to occur visibly. 9-Aminoacridine is known to be one of the few most fluorescence compounds giving a fluorescence of a violet-blue colour. Atebrin in water gives a fluorescence of green and is visible by ultra-violet light. 4
Fluorescence of a neutral acridine can be observed by adding sodium hydroxide so that ionisation is suppressed which will give an intense violet fluorescence. Due to strong fluorescence's of acridines and derivatives, these compounds are used in stain microscopy in volumetric analysis as indicators.
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Zanker has described what type of apparatus is used for acridines in measuring the fluorescence spectra. 5 Light sources is obtained from a ultra-violet lamp having an interference filter at between 365 and 546mÂµ. The light falls on to the sample which has been dissolved in ether and ethanol together as a mixture. The sample is kept in a cell at 1800C by liquidised oxygen. The Zeiss monochromator has an entrance slit on which the emitted light falls upon converted to electric current by a photomultiplier. This current is amplified by a photoelectric spectrophotometer. This is then read off as a unit of optical density.
Augmentation and Quenching
The excited singlet state lifetime can be shortened which decreases the fluorescence quantum yield. The yield can hence be increased because there are fewer molecular collisions caused by a low temperature and solvents that are less viscous.
In a strong solution, fluorescence can be suppressed. This is known as concentration quenching and can be done by:
Probability of collisions being increased
Excited and non excited molecules having different resonance structures
Dimers or micells being formed
Electronic transitions that are radiation less
Movement can lead to masking recognition of fluorescence being weak hence solutions being stronger.
An example of concentration quenching takes place in a solution of 3, 6-diaminoacridine monohyrdochloride. At 10-2m, there is no fluorescence whereas a fluorescence of an intense green is developed at 10-3m done by extensive dilution.6
Acridine fluoresces in water very strongly but in most organic liquid solvents, no fluorescence is shown.
Polarised light and Fluorescence
Polarisation from a molecule for the light given for fluorescence depends on the time taken after excitation to emit light. This is also dependent upon the relaxation time of the rotatory movement. If extensive rotation can be taken by the molecules, then there is little polarisation shown by the light of fluorescence. Polarisation can be increased by increasing the relaxtion time by the substance being placed in a viscous medium.7
Absorption spectra's can be analysed by the fluorescent spectra degree of polarisation excited by plane polarised light and can be compared with molecules such as amino acridines and serium albumin.
This type of analysis can be carried out by obtaining continuous emission using a lamp on a sample in UV light and passed through a monochromator. Wavebands can be selected and plane polarised by a prism. This can be used to cause excitation and to analyse the bands, polarisation sheets are used. Another monochromator is used to separate the beams of fluorescent waveband and the excited waveband.8
In fluorescence, there is no change of spin in the electrons. However, a small probability can occur of excitation by a change of spin in a single electron. Due to this transition, an excited triplet state is formed.
This triplet state is paramagnetic but lower in energy to the singlet excited state. This only happens in very few molecules and absorption is described as â‚¬ = < 1. Phosphorescence is the name given to the reverse of this process. The transition takes place from the triplet to the singlet excited state. Even after the beam has been excited, light continues to be emitted.
S is singlet state
T is triplet state
Bulkier substituent's that are heavier in molecular weight result to a more chance of the singlet-triplet transition occurring and this can also occur due to the structure of the substance being rigid.
To study this type of spectra, at 100 atm pressure, spectrometry in oxygen is used because oxygen is paramagnetic hence can help excite transitions in this state. In these conditions, a single - triplet absorption of 631mÂµ is shown for acridine.9
When measuring phosphorescence, a flash source that is powerful in light such as 400 watts controlled stroboscopically is targeted on to the sample usually dissolved at 1800C in an alcohol-ether mix. The light emitted is run through a monochromator followed by another monochromator. This light in a photomultiplier tube is focused using a photocathode and a galvanometer (apparatus for detecting electric current) 10 are used to read the intensity.11, 12
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Acridines in boric acid with phosphorescence have a non exponential rate decay13 and acriflavine proved this theory. Acriflavine when absorbed on silica gel and measured between 400C and -1960C gave a first order rate of decay.
Acriflavine emits 2 phosphorescence band on silica gel adsorbed occurring from the triplet electronic state.14, 15 Fluorescence spectra and can be the same sometimes as the phosphorescence spectra. E.g. Acridine Orange had a polarisation angle curve of phosphorescence in rigid sugar solution identical to acridine orange in dilute glycerol for fluorescence. The concentrations were also the same with 1 mg. per gram of quenching for both phosphorescence and fluorescence.16
Acridine orange monocation was measured in the triplet absorption at 1800C in a mix of ethanol and ether solution which resulted in 537 and 1,260 mÂµ region, showing 17 different bands. Concentration increase resulted in the decreasing of adsorption in the triplet state.17
1.2.2. Ultraviolet and visible spectra
Energy can be increased equal to absorbed energy of photon for a few moments when light is absorbed by a molecule. This results in vibrational, electronic and rotational energy. Ultraviolet and visible light produces an electronic transition whereas the other two transitions are produced by infrared light.
These spectra are used to help find the groups and find out what the molecule looks like of an unknown sample. Purity can also be determined by comparison of various spectra amongst each other.
UV spectrometry occurs when absorption takes places in the ultraviolet and visible region absorbing radiation from ground state to excited state.18 UV light causes the electronic transition to be always directionally polarised along the axis.
Acridine can undergo ionisation unlike anthracene which is similar in structure but has no heteroatom. The spectrum can be altered due to ionisation effect hence leading to wavelengths being longer which displace long wave peaks.19
1.2.3. Surface activity and association
Dimers can be observed in acridines which are in the form of ions in solution at a concentration of 10-4M. The cation of acridine at room temperature in dichloromethane can form a complex with a deep yellow colour and but is not detectable in dilute solutions.20
Surface activity has been determined of acridines in air and whilst interaction with water interface. Most acridines don't show a lower surface tension to a most extent.
The first ever accurate ionisation constant was determined in the acridine series on atebrin using various methods.
Potentiometric titration with a glass electrode
Solubility through a range of pH values
Partition between water and ether at a series of different pH values21
The strength of a base falls when the temperature is risen and if the base is stronger, the strength is lost. The high pKa value of acridine is observed due to acridine being much more soluble in alcohol than in water.
Acridine is a moderately weak base with a pKa value of 5.6 and is stronger than aniline, pyridine and quinoline that all have lower pKa values. The groups cause an effect on the acridine basic strength. E.g. an alkyl group is slightly base strengthening, whereas a nitro or a cyano is base weakening.22
When acridine was investigated, the fluorescence of acridine in an aqueous solution suggested that the pKa when in the first excited state of the cation was at 250C, 10.65.
1.2.5. Dipole moments
The dipole moment is a measure of electrical symmetry. The moment of acridine (-1.94) can be compared with the moment of pyridine (-2.21). This magnitude of a negative dipole moment shown by acridine tells us that most electrons in acridine are delocalised from the Ï€-layer towards the nitrogen atom.23
1.3. Chemical Properties
Acridine has high resonance energy and is paralleled by its great chemical stability. It can be distilled over glowing zinc and can remain unaltered when melted with potassium hydroxide.24
Electrophillic substitution of acridine can take place mainly in 2-position and less readily in the 4-position. Acridine can be nitrated which was investigated by Lehmstedt by reacting fuming nitric acid in acetic acid, giving good nitration without altering the oxidation state.25
Acridine can be chemically changed by sunlight undergoing phtoopreduction26. The reaction of photoreduction of acridine is a free radical reaction.
1.4. Biological Properties and Uses
Acridines can be used in bacterial infection, against malarial parasite, in use of anti-immune substance, in use against infections etc.
1.4.1. Use against bacterial infection
Acridine activity can be carried out on wounds without affecting the presence of serum and aminoacridines were used in the world wars to sterilise wounds without any damage caused to the wound. Aminoacridines are used in surgical work and work against the Gram-positive and Gram-negative organisms that can be found in wounds.
In the early days, mice were experimented on by in injection of either proflavine or acriflavine that are similar in structure to Acridine Orange and were cured. 160 mice were injected with a strain of streptococci and proflavine were dropped on the wounds. Out of the mice treated and controlled only 9 survived.27
1.4.2. Acridines against malarial parasite
After Atebrin was discovered, and made available, it was discovered that it was more potent in treatment of malaria than quinine hence, trials were made on native races in mental institutions.28, 29, 30, 31, 32
The standard method of suppressing was to inject the drug before the infection took place, throughout and also up to 23 days later. Bred mosquitoes, under controlled conditions, caused biting. After this the volunteers were watched for up to 40 days and if there was no sign of malaria being developed, then transfused from this patient into another. If the patients also did not have malaria, then the patient was deemed free and cured.
This research allowed scientists to work out that up to 0.6 or 0.7 grams of Atebrin suppressed malaria in P.falciparum infections (tertian malaria). Other experiments that took place allowed volunteers to take quinine, roughly the same amount of 0.7 grams daily instead of the Atebrin but anaemia and parasitaemia developed. Hence due to this, quinine is inferior in suppressing the disease.
There are however many side effects of using mepacrine. Quinine can cause ringing in the ears when taken after a meal. However, mepacrine can cause digestion problems and also adds up in the skin. After a considerate period of time, an intense yellow appearance can develop on the skin. Mepacrine does not cause liver damage or affect the uterus lining so therefore has some advantages over quinine.33, 34, 35
1.4.3. Acridines use in helminth infection
Cestode infestations can be treated by mepacrine, often a dose of 0.5 grams with 0.5 grams of sodium bicarbonate.36 This allows expulsion of entire tapeworms. This drug acts on the superficial muscles of the worm which can be eliminated in less than two hours.37, 38, 39, 40, 41, 42
Tapeworms can be more easily located in faeces because the scolex absorbs mepacrine, hence absorbing and fluorescence's as an ultraviolet light colour.43
1.4.4. Using acridines on anti immune substances
Treatment of lupus ertythemetasosus can be done with the application of mepacrine. This disease is found on the cheeks and on the bridge of the nose. This was first introduced as a cure in 1940 in Russia.44
1.4.5. Dyes and Reagents
A pigment applies to a coloured substance which never enters into the solution at any stage of application. These type of substances are mainly used in paints or as padding emulsion. A Dye is a highly coloured substance that is in solution at the time of application. Acridines have made contributions to dyes such are dyes that are soluble in water, and other in solvents.
1.5. Acridine Orange
Acridines are used as fluorescent indicators for titrations performed under ultraviolet light. These types of indicators are used when the solution to be titrated is dark or too coloured for ordinary coloured indicators to be seen. Fluorescent indicators are also used for titrations which reagents more dilute 0.1N because these indicators are brilliant and therefore little foreign is needed for introduction.45
Acridine Orange can be used to determine the degree of deterioration of wheat in storage. A strong red fluorescence is produced with fatty acids liberated from the breakdown of triglycerides.46
Acridine orange has been used for fluorescence change from yellow green of the non-ionised molecule to the orange of the cation.47
Under the fluorescent microscope, muscle is stained light blue by acridine yellow, and green by acriflavine. Acridine orange differentiates the nucleus from the cytoplasm and although it always doesn't differentiate between living and dead cells, it is useful in biopsy of treatment of cancer as a red cytoplasmic fluorescence appears signifying that the cells are deads.47
Aqueous solutions of acridine orange monocations consist only of a momomer dilute dimmer, but can form an aggregate dimmer when concentrated. Both monomer and dimmer have different absorption spectrums. The production of two or more colours by the interaction of a plant or an animal tissue is called metrachromasy. The tissue components which cause the greatest colour change in basic dyes are polymers. The fluorescence changes from green to red in aggregation.
Acridine orange has the property of staining living cells without killing them. It is toxic in excess but for purposes of vital staining only enough of the dye is used to impart fluorescence to the specimen which is placed on quartz slide under the microscope and illuminated by ultra violet light.48
Acridine Orange is also used in dyeing leather and lignocelluloses to impart a pure bright orange colour. It also used in making lakes, and as an illuminating colour in discharge printing of cotton and silk. The free base is sued in varnishes and spirit inks.
On the right is the fluorescence spectrum of Acridine orange dissolved in basic ethanol.49
Energy of incoming light is absorbed by acridine orange which is then transferred into the dye molecule. The energy does not stay within the dye forever hence is released at a different wavelength from the wavelength of the incoming light. This is why a different fluorescence colour is shown once absorbed.
Also, the ring structure allows acridine orange to absorb the incoming radiation. This energy passes around the ring. The compound is also water hating (hydrophobic due to the ring structure. In solution, the dye diffuses into membranes of surrounding micro organisms. When the acridine orange is within the cell, a complex can be formed with the DNA and RNA, affecting the wavelengths emitted.
The colour green is observed when the radiation is emitted in acridine orange-DNA complex. The colour orange is emitted in the complex of acridine orange-RNA hence the colours allow differentiation between DNA and RNA.
1.6. Literature Review on previous research
Acridine orange has been widely studied and researched by various organisations and institutions but insufficient literature has been published with respect to the analysis of acridine orange and how it is made. In this section, two articles will be discussed as they discuss how acridine can be made.
1.6.1. Microwave enhanced synthesis of acridines
Acridine was studied using microwave irradiation based on using a heat source using the Bernthsen method of synthesis. The irradiation reduced the time of formation of product and increased the yield.
Acridine could be made by heating diphenylamine with carboxylic acid using zinc chloride as the catalyst and heating at 2000C. The scientists used diphenylamine, zinc chloride and benzoic acid using different stochiometric ratio to produce the best yield of acridine formed with derivatives. It was found out that the best yield was obtained by the ratio of 1:1:1 after irradiation of 2.5 minutes.
A good way found to carry out this process was in a microwave oven which showed that making acridine in the conventional, classical way using the same ratio of compounds produced 48% acridine whereas using an oven resulted in 98% yield.50
1.6.2. Fluorescence enhancement of bis-acridine orange peptide, BAO,
upon binding to double stranded DNA
This article studied binding of double stranded DNAS to acridine orange and showed that this increased importance in cell biology and biotechnology. These scientists tried to develop a dye which could specifically show DNA sequence showing the differences between dsDNA and single stranded ssDNA.
The synthetic route in trying to develop such a dye was carried out in the following manner:
Synthetic route for BAO. (a) Sulfur, 220-230 â-¦C; (b) EtONa, CH3I, rt; (c) SPPS with Fmoc-Lys(Boc)-OH and Fmoc-Lys(Mtt)-OH, rt; (d) acetic anhydride, DCM, rt; (e) 1% TFA-DCM, rt; (f) 0.1% TFA, TIPS, DCM, rt; (g) 2, DCM, triethylamine, trifluoroethanol, 40 â-¦C; (h) 86%
TFA, thioanisole, m-cresol, rt; (i) HPLC purification.
They reported that newly synthesised BAO (bis-acridine orange) showed 200 fold more enhancement fluorescence upon binding to the dsDNA but not to ssDNA. This therefore showed that it is suitable for the specific analysis of dsDNA in the presence of ssDNA.51
There are several methods of synthesising acridine using different solvents. This includes using the A.Bernsthen synthesis, Lehmstedt-Tanasescu reaction and cyclisation of N-phenylanthranilic acid.
The method I chose to carry out was the A.Bernsthen synthesis of acridine.
In 1883, Bernsthen heated diphenylamine, benzoic acid using zinc chloride resulting in 9-phenylacridine.
Temperature used to heat varied between 2000C and 2700C, over 20 to 40 hours of heating. Lowering the temperature halved the yield.52
Tetrohydrafuran (5ml) was reacted with N, N diisopropylethylamine (5ml) into a conical flask and add 4-chloro-2-benzoic acid (0.48g) reacted with (0.5g). This was then refluxed for an hour and a half. To remove the unwanted by-product, citric acid (4cm3) with a lower pH value was added to the solution leaving the diphenylamine required.
This was reacted with formic acid (2.34g) in the presence of zinc chloride(0.8g), heated at over 200 0C to make a ring and obtain acridine with the derivatives. The nitro group was removed by reduction using HCl (25cm3) and Iron (2g).
The hydrogen on the nitrogen was then displaced by adding CH3Br. Infra red spectrums were run of starting materials and products if made.