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Synthesis of an Antiprotozoal Prodrug with Cyclopropane Structure Inhibiting the Shikimate Pathway

Info: 12832 words (51 pages) Dissertation
Published: 9th Dec 2019

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Tagged: MedicinePharmacology

ABSTRACT

Background and aim:Protozoa is a phylum comprising of single-celled microscopic eukaryotic organisms, which are associated with serious and fatal parasitic diseases such as malaria, African trypanosomiasis, leishmaniasis, and toxoplasmosis. Nevertheless, due to resistance towards traditional antimalarial medications and a new mutated strain of Plasmodium falciparum protozoan in Vietnam, treatments are limited.  There is an urgent need for a new antimalarial drug associated with a new mode of action with minimum toxic side effects. There has been a great interest in synthesising potential medications inhibiting the shikimate pathway.

The shikimate pathway is an essential biosynthetic route for plants, bacteria, fungi and protozoa to synthesise aromatic pre-cursors of tannins, flavonoids and vitamin K.  Since this pathway is absent in mammals hence inhibiting this pathway will not cause undesirable side effects.

The aim of this project was to synthesise a potential antiprotozoal pro-drug inhibiting the shikimate pathway in protozoan.  The drug design associates by mimicking the structure of shikimic acid which, is the fifth intermediate in the pathway. However, the pro-drug will inhibit the seventh enzyme in the shikimate pathway.

Methods:  The experiment consists of four stages to synthesis the desired drug. Shikimic was firstly acetylated, the carboxylic acid was reduced, cyclopropanation and followed by oxidation process to reform the carboxylic group. Each product was analysed by various analytical techniques such as TLC, NMR, IR and mass spectroscopy.

Results: The first step of the reaction was repeated eight times to obtain the desired intermediate with better yield, higher mass and purer product. Each reaction was critically analysed and modified. The last combination reaction gave a yield of 89% and 1.60g. Step two, method was modified to use distilled THF instead of digylyme to reduce the carboxylic acid group. Some traces of starting material were still present in the intermediate product. This intermediate had a yield of 64% .

Conclusions and Implications: The first two intermediates were successfully formed in the synthetic route. The first reaction can be optimised by stirring the reaction for two hours. Removing pyridine with 1M HCl (4x20ml) and using copper sulphate for visual indication. Also using distilled pyridine and acetic anhydride bottles reagents.  Step two, optimised the reaction by using distilled THF instead of digylyme which gave promising analytical results.

Keywords: shikimic acid, shikimic pathway, protozoan, inhibitor, cyclopropanation, and acetylated.

ABBREVIATIONS

 

b.p                                          boiling point

bs                                           broad singulet

Brine                                      sodium chloride concentrated in water

COSY                                     correlation spectroscopy

d                                             doublet

dd                                           doublet of doublet

DEPT                                     distortionless enhancement by polarization transfer

Diglyme                                  bis(2-methoxyethyl) ether

E. Coli                                     Escherichia coli

ET2O                                      diethyl ether

FT                                           Fourier-transform

FTIR                                       Fourier-transform infrared spectroscopy

HSQC                                     the heteronuclear single quantum coherence

Hz                                           hertz

J                                              coupling constant given in Hz

m                                            multiplet

MHz                                        megahertz

MS                                          mass spectroscopy

NADH                                     Nicotinamide adenine dinucleotide

P. falciparum                          plasmodium falciparum

Pi                                            phosphate

pK                                          dissociation constant

®                                            register

Rf                                           retention factor

s                                             singulet

δH                                                               (MHz, solvent) 1H data

δc                                                               (MHz, solvent) 13C data

t                                              triplet

TM                                          trademark

TMS                                        tetramethylsilane

UV                                           Ultraviolet

SA

ca

CONTENTS

 

  1. INTRODUCTION………………………………………………………………. 6
    1. Protozoan disease ………………….……………………………………. 6
    2. Malaria………………………………..…………………………………… 6
    3. Shikimate Pathway  ………………………………………………………. 7
      1.     Importance of Essential Amino Acids in Humans………………. 7
      2.     3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate (DAHP)……..  7
      3.     3-Dehydroquinate (DHQ) ……………………………………………. 8
      4.     Shikimate …………………………….……………………………….. 8
      5.     Shikimate 3-phosphate ….……………………………………………. 9
      6.     5-Enolpyruvylshikimate 3-Phosphate (EPSP) ………………………
      7.     Chorismate …………………………………………………………….
    4. Shikimic Acid Derivatives ………………………………………………..
    5. Shikimate pathway inhibitor…………………………………………….
      1.     Tuberculosis ………………………………………………..……… 9

1.5.2   Malaria

  1. Drug Design of Potential Prodrug Targeting Protozoa…………  11
    1.     Inhibitors mechanism……………………………………………. 13
  2. Development of a possible inhibitor derived from shikimic acid……… 15
  1. AIMS AND OBJECTIVES………………………………………………….. 20
  2. EXPERIMENTAL……………………………………………………………. 21
    1. Instruments ………………………………………………………………… 21
    2. Chemicals
    3. Analytical techniques
    4. Thin Layer Chromatography (TLC)
    5. Liquid Chromatography-Mass Spectrometry (LC-MS)
    6. Infrared (IR) Spectroscopy
    7. Nuclear Magnetic resonance spectra (NMR)……………………………………………………                                          21
  1. Methods ……………………………………………………………………  22
    1.  Acetylation of shikimic acid

4.2 Synthesis of 5-(hydroxymethyl)cyclohex-4-ene-1,2,3-triyl triacetate

  1. RESULTS AND DISCUSSION…………………………………………….. 24
    1. Synthesis of (3R,4S,5R)-tri-acetoxy-1-cyclohexene-1-carboxylic acid 12 from shikimic acid…………………………………………………….                            24
    2. Synthesis of 5-(hydroxymethyl)cyclohex-4-ene-1,2,3-triyl triacetate…. 37
  2. CONCLUSION……………………………………………………………….. 49
  3. FUTURE WORK……………………………………………………………..  50
  4. REFERENCES………………………………………………………………. 51
  5. APPENDICES………………………………………………………………..  54
  1. INTRODUCTION

1.1 Protozoan Diseases

Protozoa is a phylum comprising of single-celled microscopic eukaryotic organisms, which are classified under the Kingdom of Protista. These eukaryotic species are highly abundant in moist and aquatic environments and include amoebas, flagellates, ciliates, sporozoans and many other forms. In nature, they are found in the form of free-living organisms or as parasites within other organisms. (1) Protozoan infections are parasitic diseases, where the parasites invade a human host such as Plasmodium falciparum. Malaria is a life-threatening condition with a mortality rate of more than a million in tropical regions of the world.(2) The mortality rate of this particular disease is still high despite of existing traditional medications. A study focused on number of years reduced due to ill heath associated with the four most common parasitic diseases include: malaria (protozoa: Plasmodium, P), African trypanosomiasis (Trypanosoma), leishmaniasis (leishmania) and toxoplasmosis (toxoplasmosis).  Malaria had the highest figure of disability adjusted life year.

This is due to poor adherence, expensive medications, resistance occurring towards P. falciparum strain, adverse side effects or a lack of the availability of a licensed vaccine. Hence, research is currently being undertaken to produce new effective antiprotozoal drugs.(1,2)

1.2 Malaria  

Malaria is a blood borne disease spread by an infected Anopheles mosquito. It is associated with high grade fever. The disease is caused by various species of plasmodium, commonly P. falciparum which invades the human body via inoculation of sporozoites into the blood from a female Anopheles, as demonstrated in Figure 1.

Malaria is found in the areas of Middle East, Africa, the Caribbean, the parts of the Central and the South of America, Southeast and South of Asia. It affects approximately half of the world’s population.(3) Moreover, Malaria is a cause of further concern as its enormity increases by gradually spreading up north due to global warming.(4) In 2016, there were 440,000 deaths reported out of 216 million cases of malaria. Approximately two-thirds of this mortality figure represented children under the age of five. (5)

Figure 1. Explains the life cycle of Malaria. (6)

1.3 New Strain of Malaria

A new strain of the Plasmodium falciparum parasite is spreading in South Vietnam due to mutation, which cannot be killed by dihydroartemisinin (DHA)-piperaquine, also called ‘super resistant malaria.’ DHA is the first line antimalarial medicines against Plasmodium falciparum, in Vietnam.(8) The spread began in western Cambodia moved to north-eastern Thailand and into Southern Vietnam.  Thus, new antimalarial medications are required to target the new super resistant malaria with a new mode of action. This conclusion was supported by subsequent work since 2004, which mentioned resistance exists towards all class of anti-malarial medications e.g. chloroquine, proguanil, malarone, doxycycline and mefloquine.(9)

1.3 Shikimate Pathway

Protozoa along with bacteria and plants commonly use one of the major biosynthetic routes, known as the shikimate pathway for the synthesis of essential aromatic amino acids (L-tyrosine, L-tryptophan, and phenylalanine) from simple precursors. Tall plants use these amino acids to form secondary metabolites such as tannins, lignin, flavonoids, and alkaloids.

This pathway is not present in mammals hence it will not cause any toxic effects in them. Therefore, if a new enzyme inhibitor of this pathway is synthesised, it can potentially be targeted against ‘super resistant malaria.’

The pathway was discovered by David Sprinson et al as a biosynthetic route for amino acids production from simple building block precursors, via seven metabolic steps to form chorismate. It is present in both eukaryotic and prokaryotic organisms, which produce matching intermediates and end products. However, the primary structure between the two is different, because of various enzymes found in shikimate pathway. (11,12)

 

  1.     3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate (DAHP)

Figure 2.  PEP and E4P undergo condensation process and catalysed by DAHP synthase forming 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate (DAHP).

The initial step of the shikimate pathway involves the reaction being catalysed by DAHP synthase with Erythrose 4-phosphate (E4P) and Phospho-enol-pyruvate (PEP), to form six-membered heterocyclic structure. This is summarised in Figure 2. This enzyme consists of three aromatic amino acid rings which are refined by electrophoretic homogeneity process from various microbial sources and vegetables to obtain pure enzymes. This enzyme is activated by a metal ion e.g. Manganese (Mn2+) and reduced thioredoxin.  This conclusion is supported by a study that experimented on Escherichia coli (E. coli) and further discovered that the DAHP synthase consisted of three isoenzymes which can inhibit the production of phenylalanine, tyrosine, and tryptophan. Both PEP and E-4P are derived from glycolysis process. (11,14)

1.3.3 3-Dehydroquinate (DHQ)

The second metabolic step involves, the catalytic reaction of DAHP with DHQ synthase  to synthesise 3-dehydroquinate (DHQ) by removal of a phosphate group, as demonstrated in Figure 4. In E.coli cell, DHQ synthase enzyme needs divalent cations for activity and is activated by inorganic phosphates such as Co2+ and NAD+. The cyclic ring is formed during this stage, before undergoing five further stages, namely: alcohol oxidation, phosphate β-elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Since this enzyme plays an essential role in the synthesis of amino acids, it can potentially be used to inhibit the growth of Helicobacter pylori disease. (11,14)

  1.    3- Dehydroshikimate

Step three involves 3-Dehydroquinate Dehydratase (DHD) enzyme which is found in bacteria and exists in two forms. Dehydrating DHQ involves two types of reactions, both of which undergo the same process and produce the same product. Table 1 summarises the differences between type 1 and type 2 of dehydroquinase. (15)

 

Figure 4. DHQ is catalysed by 3-Dehydroquinate Dehydratase (DHD) to dehydrate the molecule to form 3-Dehydroshikimate.

Table 1. The summary of the differences between type 1 and type 2 of dehydroquinase.

Type 1 Enzyme Type 2 Enzyme
  1. Unstable during heat
  2. The mechanism involves a cis-elimination
  3. Type of reaction: anabolic in the shikimate pathway
  4. Consist of lower Km value, suggesting it consists of higher affinity since less substrate concentration is required compared to type two to reach half of Vmax
  5. Commonly found in plants and bacteria
  1. Stable during heat
  2. The mechanism involves the trans- elimination
  3. Type of reaction: catabolic in the quinate pathway

1.3.4  Shikimate

Figure 5. 3-Dehydroshikimate is reduced using Shikimate dehydrogenase to form Shikimate.

To form

Is reduced t

Analysing E. coli bacterial biochemistry showed that the fourth step in this pathway involved DHS being reduced to shikimate whilst being catalysed by NADP-dependent shikimate dehydrogenase (SDH). However, in plants, step three and four of shikimate pathway reaction is carried out by a bifunctional enzyme DHD/SDH. This process ensures that the reaction is completed, and the accumulation of the unreacted substances is prevented e.g. dehydroshikimate. (11,14)

1.3.5 Shikimate 3-phosphate

Figure 6. Shikimate is catalysed by shikimate kinase (SHK), which involves ATP-dependent phosphorylation to form Shikimate 3-phosphate (S3P).

The fifth step of this pathway is associated with a phosphate group from ATP transferred to the 3-hydroxyl group on shikimate. Therefore, shikimate kinase consists of two active binding sites for ATP and shikimate, the result of which is two products: ADP and shikimate 3-phosphate. Shikimate kinase is commonly found in flowers but less so in leaves and roots.

In E. coli, there are two isoenzymes of shikimate kinase. Since Isoenzyme 2 has 100 times lower Km value it is used in the shikimate pathway. It has been studied in spinach and tomatoes. (11,14)

  1.     Enolpyruvylshikimate 3-Phosphate (EPSP)

Figure 7. S3P being catalysed by EPSPs to form EPSP.

The sixth step involves enzyme,  5-Enolpyruvylshikimate 3-Phosphate Synthase (EPSPs), catalysing S3P to form Enolpyruvylshikimate 3-Phosphate(EPSP).

A second enolpyruvyl is derived from PEP to bind onto the second hydroxyl group of shikimate-3-phosphate.

  1.     Chorismate

Figure 8. Demonstrates the mechanism of action of EPSP being catalysed by chorismate synthase (CS) to form chorismate.

The last enzyme in this pathway is chorismate synthase (CS) which aids in eliminating the phosphate from EPSPS to form chorismate. In addition, two double bonds are formed in the benzene ring. Chorismate is activated by the presence of a reduced flavin nucleotide (FMNH2) despite the overall reaction being redox neutral. Figure 9 explains the mechanism of (17) this main precursor to form both primary and secondary compounds as is illustrated.

  • Tetrahydrofolate (vitamin B9) via Aminodeoxychorimate synthase
  • Salicylate via Isochorismate Synthase
  • Phylloquinone (Vitamin K)
  • Folate via ADC synthase
  • Ubiquinone via Chorismate lyase
  • Folate ADC Synthase

Chorismate

Anthranilate synthase

 form Anthranilate

Chorismate mutase form Prephenate

L-Tyrosine

Major classes of secondary metabolites:

  • Phenylpropanoids
  • Tocochromanols (vitamin E)
  • Isoquinoline alkaloids
  • Glucosinolates

L-Tryptophan

Major classes of secondary metabolites:

  • Glucosinolates
  • Indole alkaloids auxin
  • Camalexin

 

L-Phenylalanine

Major classes of secondary metabolites:

  • Phenylpropanoids
  • Glucosinolates
  • 2-phenylethanol

Figure 9. Primary and secondary metabolites formed with the specified type of enzymes.

1.4 Shikimic Acid Derivatives

Shikimic acid (SA) is named after an oriental plant called ‘shikimi-no-ki’ in Japanese and is known by its anionic form ‘shikimate’. It was isolated in 1885 from the fruit of Illicium religiosum by Eykman, who defined shikimic acid as a ‘trihydroxy-cyclohexene carboxylic acid.’

However, the stereochemistry was recognised in the 1930s by ‘Fischer, Freudenberg, and Karrer.’  Another name of shikimic acid is shikimate, in its anionic form, which is an essential intermediate biochemical metabolite in the shikimate pathway. The pathway has played a key role in the pharmaceutical industry to synthesise inhibitors from SA to produce new antibacterial, antiviral and anti-parasitic drugs. SA possess several unique characteristics and its usages include it being employed as anti-inflammatory, antioxidant, antithrombotic, anticoagulant and analgesic agent. SA plays a dual role as a crucial element in medical chemistry as well as being a starter product in organic synthesis.

SA is also obtained by Diel Alder reaction and microbial fermentation from glucose using recombinant E. coli. For example, viral infection e.g. influenza causes mortality rate of ca. 500,000 and affecting approximately ‘20% of the worldwide population.’

SA has been used as an intermediate precursor to produce (a neuraminidase inhibitor) ‘antiviral drug oseltamivir (Tamiflu®) as is depicted in Figure 10.’ This process is completed in nine steps and has proven to be very efficient. This medication is used to treat influenza virus types A and B, by blocking the neuraminidase enzymes which halts the virus from spreading. It is very effective in treating two-week-old babies and elderly.

Moreover, due to the increase in resistance, an alternative to oseltamivir, peramivir (RapiactaTM), was produced. Another derivative of SA is Zeylenone, which is synthesised initially by the stereoselective process. This derivative has shown antibiotic, antiviral and anticancer effects. However, it is mainly used for treating cancer. Fluorinated analogues such as (6S)-6-fluoro-shikimic acid, are synthesised due to antibacterial activity against several E. coli strains. (18)

(2)

(3)

(4)

(1)

(2)

(1)

(5)

Figure 10. SA used as an intermediate precursor to produce (a neuraminidase inhibitor) ‘antiviral drug oseltamivir (Tamiflu®) and other derivatives.

1.5 Shikimate pathway inhibitors

1.5.1 Herbicide

The seven enzymes and its intermediates in shikimate pathway have been potential targets to synthesise a new anti-microbial, herbicides and anti-parasitic drugs. inhibiting this pathway will prevent the growth of the cell and will not cause toxic side effects in humans.  In 1974 a  broad-spectrum herbicide called Roundup® was synthesised. This was the first commercial product to inhibit the shikimate pathway.

The active ingredient in this herbicide is  glyphosate. It acts as a competitive reversible inhibitor and halts the pathway by mimicking an analogue of EPSP synthase enzyme (Fig 8). This prevents shikimate 3- phosphate from binding on to the active site of EPSP enzyme, leading to inhibition of further protein synthesis.4

Since shikimate pathway is absent in animals, this makes Roundup® a safe herbicide to be used. Roundup® has been modified to ‘Roundup Ready (Ultimax).’ This liquid is sprayed onto the crops which contain ‘glyphosate-resistant crop plant’. The difference between the two herbicide is that with the modified version, weed is killed without affecting crop plants.(11,14,16)  However, this effect would lead to deficiency   of essential amino acids in human’s diet. This effect will lead to depression, insomnia, unstable moods and lacking vitamin B3

1.5.1 Tuberculosis

Shikimate kinase enzyme has been inhibited previously to treat Tuberculosis, which is a bacterial infection caused by a tubercle bacillus (Mycobacterium tuberculosis), commonly affecting the lungs, abdomen, bones, and glands. The bacteria slowly divide and spread across the lungs to form tubercles or hard nodules. This causes respiratory tissues to be broken down and affects the blood vessels, producing cavities in the lung. The current treatment that is taken for six months, involves two antibiotics: isoniazid and rifampicin.  However, the increasing drug resistance (XDR-TB) makes the current medication less effective in targeting the bacillus. However, the Shikimate pathway is present in tuberculosis bacteria in which studies have demonstrated the inhibition of the fifth enzyme associated with this pathway, namely shikimate kinase (MtSK), which prevents the growth of the bacteria, leading to cell death. Furthermore, the shikimate kinase inhibitor would not cause any toxic and undesirable effects in humans as the pathway is absent in them. (19)

1.6.2 Malaria

Previously, a study was carried out, which demonstrated the inhibition of the formation of chorismate while targeting P. falciparum. This prevented the spread of malaria. The study focused on three different structures and concluded (6R)-6-fluoro-shikimic acid to be the most effective inhibitory substrate as its structure mimicked shikimic acid.

(6)

Several studies have discovered that many parasites are sensitive to glyphosate (N-(phosphonomethyl) glycine) with a concentration range of 1-6mM. Thus, shikimate analogues may be the universal inhibitors of other protozoan parasite diseases such as toxoplasmosis and cryptosporidiosis. (20)

  1. Drug Design of Potential Prodrug Targeting Protozoa

The potential new antiprotozoal pro-drug mimics shikimic acid because of its similar bond length (difference of 5 pm), it has a complementary structure to shikimate kinase enzyme. Hence, inhibiting the conversion of shikimic acid into shikimate-3- phosphate will result in inhibiting protozoa proliferation.

The structure 8 consists of carboxylic acid group, three hydroxyl groups, and a cyclopropane conformation. The only difference between the two structures, as illustrated above, is the alkene and cyclopropane conformation. The main inhibitor property is derived from the cyclopropane structure, which consists of the same half-chair conformation structure and gives similar chemistry to C=C bond once the ring is interrupted. Therefore, it will be effective in preventing the proliferation of some protozoa, bacteria and fungi. (21)

Once the shikimic acid analogue binds onto shikimate kinase substrate, the reaction is still catalysed. In the next step of shikimate pathway, the product is catalysed by EPSP synthase enzyme, converting 5-OH to S3P. This is the same step as witnessed earlier in shikimate pathway. However, the subsequent step involves phosphate cleavage at C-5, aided by Chorismate synthase enzyme, forming a radical adjacent to cyclopropane.

This is because the shikimic acid analogue prevents the normal activity of CS of reducing flavin mononucleotide (FMN). This effect causes the opening of the cyclopropane, producing a more stable ring consisting of seven carbons attached, at C-1, to a second radical, which attaches onto the active site of CS and causes irreversible inactivation of this enzyme due formation of a covalent bond. This effect terminates the synthesis of both primary and secondary metabolites in super resistant malaria from growing further. (21)

 

Figure 10. Illustrates a possible mechanism of action of inhibiting Chorismate synthase.

To convert shikimic acid to a structure consisting of a cyclopropane, Simmons-Smith reaction can be used. This involves forming a zinc carbenoid which leads to a carbine. This compound attacks the double bond in the cyclohexene, forming a cyclopropane group, as demonstrated in figure 11. (22,23)

Figure 11. Demonstrates reduced shikimic acid analogue undergoing Simmons-Smith reaction to convert the alkene bond to a cyclopropane group.

Cyclopropane analogue of shikimic acid

Figure 12. Curly arrow mechanism demonstrates the Simmons-Smith reaction for the formation of the cyclopropane analogue of shikimic acid

  1. Aim and Objectives

The aim of this project is to produce a potential inhibitor of cyclopropane from shikimic acid to bind onto shikimate kinase enzyme. However, it forms an irreversible bond in chorismate synthase enzyme, so that the shikimic pathway will be halted leading to the development of a new potential antiprotozoal drug.

 

Objectives:

There are four steps involved in the synthesis of a successful inhibitor against chorismate synthase enzyme and the following is an overview of the steps involved in the laboratory:

Each product obtained will be analysed using; nuclear magnetic resonance (NMR) and infrared spectroscopy (IR), mass spectroscopy and thin layer chromatography.

Cyclopropanation

Reduction

Acetylation

(1)

(9)

(5)

(10)

Oxidation

(7)

Figure 13. An overview of the steps involved in the synthesis of an inhibitor, a potential prodrug, against the shikimate kinase enzyme

  1. EXPERIMENTAL

 

  1. Instruments

The following equipment was used during the labs to carry out this experiment:

25ml, 50ml, 100ml and 250ml of round bottom flasks; 10ml and 100ml of measuring cylinder; 50ml and 250ml of beaker; reflux condenser and tubing; stirring mantle; nitrogen bubbler; guard tube; magnetic stirrer; spatula; separating funnel; glass rod; sample vials; funnel; TLC tubes and rotary evaporator.

  1. Chemicals

Majority of the reagents that were used in the experimental procedure were obtained from arros such as borane tetrahydrofuran complex solution 1.0M in THF (99%), distilled pyridine, and 1g of shikimic acid ≥99% from  sigma- aldrich

To minimise the cost 5g of shikimic acid was later ordered from arros.

Other chemicals and reagents were readily available in the laboratory: acetic anhydride, pyridine, magnesium chloride, ethyl acetate, toluene, hydrochloric acid, potassium chloride, dichloromethane, chloroform, magnesium sulphate.

  1. Analytical Techniques

In this experiment four analytical techniques were used to examine the synthesised product by purifying and isolating the structure using: thin layer chromatography (TLC), Liquid chromatography-mass spectrometry (LC-MS), infrared spectroscopy (IR) and nuclear magnetic resonance (NMR) spectroscopy). The values obtained were compared against literature figures to confirm the new desired product.

  1.     Thin Layer Chromatography (TLC)

This technique involves two phases: the mobile phase and stationary phase. Three different respective mobile ratios of toluene and ethyl acetate were used including 2:1, 1:2, 3:2. Toluene was chosen due to its polar solvent property and used in high concentration to match the polarity of the synthesised product. Ethyl acetate is a non- polar solvent.

The stationary phase consists of silica gel plate which comprises of polar groups (Si-OH). This enables the mobile phase eluent as it rises upwards in the capillary, separating the products. This pattern can be detected by using UV light and visual analyses can be further achieved by staining the TLC plate with potassium permanganate (KMnO4), which is an oxidising agent, forming the following ions K+ and MnO4.   The MnO4 ion is the key ion which interacts with the secondary alcohols, forming an unstable intermediate product, which is seen as a yellow streak on the TLC plate. The Rf values are calculated and compared to the literature value.

3.3.2  GC-MS Spectroscopy

The gas chromatography- mass- spectrometry (GC-MS) technique was used to confirm the structure of the desired intermediate by analysing its different fragments. The sample was prepared in 2mg in 1000ml of hexane and acetone and injected into GC-MS, which passed through the column and separated the products according to their polarity.

3.3.3 Infrared (IR) Spectroscopy

Presents a spectrum with distinctive peaks that illustrates different functional groups existing in the product. This technique confirms the structure of the desired product. This is achieved by each chemical bond absorbing infrared at a distinctive wavelength. The concentrated oil was placed between the two KBR plates.

3.3.4  Nuclear Magnetic resonance spectra (NMR)

This technique was used to analyse and identify both proton and carbon chemical environments in the synthesised product. 20mg was measured from the product and dissolved in chloroform-d (CDCl3) to prepare a 4.8cm sample in an NMR tube. The sample was previously filtered using cotton wool to form a suspension free sample before running in Bruker Avance lll 400 two-channel FT-NMR spectrometer and analysed using spinworks.

3.4 Method

3.4.1  (3R,4S, 5R)- Triacetoxyl-1-cylohexene-1-carboxylic acid (5)

(5)

Shikimic acid was measured 0.4 g (2.3mmol) in a weighing boat and placed into a 100ml bottom flask. Pyridine/Acetic anhydride (2:1; 9ml) were added to dissolve the solute. This was stirred for five hours under the vacuum. The mixture was then evaporated to become concentrated using a rotary evaporator, followed by the addition of toluene to remove pyridine by co-evaporation. Ethyl acetate was then added to the residue and washed sequentially with hydrochloric acid 1M (10mlX3) and brine (5mlX3). The organic layer was obtained, dried with MgSO4, and evaporated. The residue was purified using flash chromatography. (Tol: EA: Acetic acid; 3:1:0.5%) to form the compound (0.42g, 1.40 mmol) as a colourless oil in 82% yield. Rf=0.27 (Tol: EA; 2:1). (24)

 

1H NMR (400MHz, CDCl3) δH 9.29 (bs, 1H, COOH), 6.87 (m, 1H, H-2), 5.76 (m, 1H, H-4), 5.29 (m, 2H, H-3, H-5), 2.91 (dd, 1H, J= 18.66, H-6´), 2.45 (dd, 1H, J= 18.66, H-6), 2.09, 2.08, 2.07 (3s, 9H, 3OC(O)CH3).

13C NMR (400MHz, CDCl3) δC 170.43 (COOH), 170.01, 169.93 (3 OCOCH3), 135.12 (C-2), 130.61 (C-1), 67.46, 66.74 (C-4, C-5), 66.01 (C-3), 28.04 (C-6), 20.99, 20.74, 20.73 (3OCH3).(24)

4.2   5-(Hydrooxymethyl) cyclohex-4-ene-1,2,3-triyl triacetate

Initially, 1.368g of the previous compound formed was weighed and dissolved in 20mL of dry diglyme, at 0ºC under an inert atmosphere of N2, with borane tetrahydrofuran complex solution 1.0 M in THF (8ml). Then the formed complex solution was added drop by drop and stirred 48 hours. Thereafter, 150ml of water was added and extraction with chloroform procedure was performed. The mixture was washed by aqueous sodium bicarbonate and followed by water. The organic phase was dried with MgSO4 and subsequent evaporation, the compound (13) was obtained as a pink oil.(25)Rf=0.1 cm.

(9)

 

RESULTS AND DISCUSSION

Formation of acetyl  groups for alcohol protection

In order to synthesis compound 5, a modified synthetic method was carried out by Carbain et al in 2010..

Figure 13. Formation of (3R,4S,5R)-tri-acetoxy-1-cyclohexene-1-carboxylic acid, 5from shikimic acid.

Acetylation process involved dissolving shikimic acid in pyridine and acetic anhydride whilst stirred over certain hours (1h, 2h, 48h, and 72 hours).  It is important to protect the alcohol groups to prevent them from being oxidised at the fifth step of the reaction. This reaction involves four steps as demonstrated in figure 14. Carbain’s et al  methodology of this step of reaction was particularly selected. Due to previous study reported higher yield of 88% product using pyridine, compared to using perchloric acid and acetic acid, which provided a yield of 54%.

Pyridine is described as heterocyclic aromatic organic compound with a high boiling point (115 0C). Carbain et al methodology mentioned to add toluene to remove pyridine by forming an azeotropic mixture. This new product is more easily evaporated from the mixture due to a lower boiling point (110.5 0C). Followed by extracting the mixture with ethyl acetate, creating the organic layer and washed with 1M HCl and brine water. Next, the top organic layer in separating funnel was dried with MgSO4 and concentrated by co-evaporation with chloroform ten times. This ensured that the residue was removed. This proposed methodology by Carbain et al was explored in eight different methods due to optimising the yield and purity of structure 5.  It is essential to obtain sufficient amount of yield and mass, in order to reach to the fifth step of the reaction with a pure product.

Optimum Procedure Method 6

A higher yield and a purer structure 5 was obtained from this particular method.

This was achieved by firstly distilling pyridine and acetic anhydride reagents before use. However  a better laboratory bottle of acetic anhydride was used. Three experiments were ran simulatenously to mimic the same conditions. 400mg of shikimic acid was dissolved in pyridine/ acetic anhydride (2:1; 9ml) for two hours. TLC was used to calculate the Rf values comparing all three experiments against the starting material. All three experiments confirmed acylation process and had the same Rf value (0.2 cm). Afterwards all the mixtures was placed into a 250ml bottm flask. The extrcation process was repated couple times to gain maximum yeild. This involved mixing the residue with 30ml of 1M HCl. Then 120ml of ethyl acetate was added, followed by washing with 1M HCl (3x 30ml) and brine water (5mlx3).  Cupper sulfate was added to give a visual indicationof complete  removal of pyridine from the organic layer.  The residue was dried with MgSO4 and concenatrated ten times with chlroforom under vaccum. This ensured ethyl acetate, an unreactive solvent was sucessfully removed.  This product was further analysed by various anatlytical tecniques which confirmed the synthesis of the desired product with minimum impurities. Hence this intermediate with highest yeild of 89% (1.6049g) was carried forward to the second step.

 The progress of the reaction was analysed by TLC plate. Carbain’s et al methodology can be optimised.  Pyridine was more effectively removed by addition of 1M HCl before mixing it with ethyl acetate This is because HCl protonates the pyridine producing pyridinium chloride, which is easily extracted by dissolving into the aqueous layer in the separating funnel (Fig 14.).  Also using copper sulfate and sodium bicarbonate enhanced the extraction process.

Figure 14. Pyridine reacting with HCl to form pyridinium chloride salt.

This product was further analysed by various anatlytical tecniques which confirmed the synthesis of the desired product.

1H NMR Spectrum

The 1H NMR spectrum confirmed successful synthesis of structure 5. In fig..  shows three peaks at 2.09, 2.08 and 2.06 ppm representing the three methyl groups from the acetoxy groups (CH3). Each peak had an integration of three protons, matching with the literature values. Since there is no hydrogen to protonate the adjacent carbon a singlet of multiplicity was observed. There were two spots on TLC plate, suggesting a side product is formed. However, the integration ratio of the impurities to the methyl group is 1:3, respectively. Therefore, the impurities are very small hence, this intermediate was further reacted in step two of the synthetic route. The methylene group with two hydrogens at H-6 position gave doublet of doublets peak at 2.91 and 2.44 ppm.  The coupling constant of these two points was 18.3Hz. The broadest peak in the spectrum appeared at 8.77 ppm which was the hydrogen protonated from the carboxylic acid. The proton environment from H-2 to H-5 appeared in the spectrum as multiplet at 6.87 (H-2), 5.76 (H-3) and 5.3(H-4, H-5). The coupling between these protons were analysed by the COSY spectrum.

COSY Spectrum

Figure.. further confimes the synthesis of the desired product by looking at the coupling in the ring protons. There was no coupling between H-9-11 because there were no hydrogens protonating its adjacent carbon.   Coupling is noticed between proton  H-2,with H-3. Both protons of H-6 and H-7 are coupled with eachother whlist proton H-6, is coupled further with H-2, to H-5. A distinctive coupling exist between proton H-3 to H-5.

DEPT- Q NMR Spectrum

All seven peaks appeared in the spectrum. The carbon in acetoxy group (O(C)OCH3) and carboxylic acid group both appeared as negative peaks at 170.013, 169.928 and 169.919 ppm. This is because there was no protonation of hydrogen on adjacent carbon. However, the ring carbons C-2 to C- 5 all showed positive signals at 135.04 (C-2), 67.471 (C-4), 66.750 (C-5), 66.07 (C-3). This is because of all the carbons are attached to one protonating hydrogen (CH).  Whereas C-1 and C-6 showed a negative peak at 130.73, 28.074 ppm, respectively. The attached protected groups were further analysed by the HSQC (fig) confirming the desired structure of 5 synthesis.

IR spectrum

Comparing the IR spectrum of the optimised method to shikimic acid, confirms synthesis of 5. There is a  new broad weak peak of carboxylic acid group (COO-H) at 3500-2500 cm1 region.  There is less peaks compared to shikimic acid at 1300-1000 cm-1 region showing the O-H bond from the alcohol groups. Moreover, another difference between the two spectrums is that in CR spectrum there is no peak seen at 3217.03 cm-1 which represents the O-H group as free non-H bonding (fig). This is due to structure 5 no longer consuming alcohol groups since they have been acetylated.  These groups are further analysed by mass spectroscopy see table..

Ultraviolet Spectrum 

However, structure 5 had a pale pink colour, consisting of chromophore. This is an unsaturated molecule with a covalent bond which absorbs light of a spcific wavelength (4.16 nm). In Fig..   some regions of the molecule are highlighted suggesting possiblity of where chromophore can be present. The colour that is reflected is not absorbed by ultraviolet spectrum.

There were five other procedures used  to carryout actylisation process on shikimic acid. Each method was constantly analysed and modified to obtain higher yeild, mass  and a pourer product.

Method 1

Carbain’s et al method was explored. The two regaents: pyridine and acetic anhydride were not distilled until the optimum 6th method . However previous work on this projet (P.Augilar) gained low yeild comapred to Carbain et al study. This became difficult for her to go beyond step two in the sythetic route. Therfore to avoid this problem the regants and solutes were all scaled up. Hence 600mg (3.45 mmoles) of shikimc acid was dissolved in acteic anydride and pyrdine (1:2, 13.5ml). TLC anaylysis after 72 hours, showed pyridine was still present despite of being concentrated with  toluene. This concludes that an azeotropic mixture was not efficient in removing pyridine. This reagent was nevertheless removed effectively by washing the organic layer with 1M HCl (20mlX3).  Low yield of 54% was obtained.

The 1H NMR spectrum (Fig) shows the methyl groups (H-9 to H-11) at 2.10, 2.09, 2.07 at a deshielded location. The three peaks are integrated for six protons instead of nine protons. Therefore, the reaction was not successful. However, TLC plate showed one clear spot thus flash column chromatography technique could not be carried out. The product consists of lots of impurities and hence cannot proceed to the second step of the reaction.

Method 2

Previous method was modified to exclude impurities by running the reaction for 48 hours. 600mg of shikimic acid was used in pyridine and acetic anhydride (1:2; 13.5ml). a higher yield was obtained 56%. The sample was not concentrated with toluene since pyridine was successfully removed  with 1M HCl (20mlx5).  The TLC plate was analysed under UV light to check if pyridine is still present. Then the plate was dipped into KMnO4  dye a yellow spot appeared, confirming the presence of secondary alcohol structures. Rf 0.25( Tol:EA; 2:1).

Overall structure 5 was synthesised but 1H NMR spectrum showed lots of impurities.  The three singlet peaks of methyl from acetyl groups are shown in the desired region. However, there are other two equal ration of impurities. The proton integration of all the three peaks is 6. Moreover, the IR spectrum is different to shikimic acid, confirming the absence of alcohol groups. There is no longer a stronger peak at 2880.15 cm-1.  The impurities could derived from the rotary evaporator and the reaction was left for too long.

Method 3

The aim of this experiment was  to find the optimum rate of the reaction to obtain the highest yield and purer product. The method was further modified by shorting the rate of the reaction to two hours.  This is because the longer the duration of the reaction the higher the chance of impurities forming. 600 mg of shikimic acid was dissolved in pyridine and acetic anhydride (2:1, 13.5ml). The 1H NMR, 13C NMR and IR gave promising results with a yield of 66%. Rf 0.26 (Tol:EA; 2:1).  However, the proton integration of the three methyl groups was less than expected (8 protons was seen). The IR spectrum looked very similar to the previous method. Hence, with shorter rate of reactions the product contains less impurities.

Method 4

The purity of the product was explored further by reacting the reaction over one hour. However, the analytical data showed some starting materials being present with the product. The goal of this experiment was to optimise the mass and purity of the product.The same method was repeated apart from leaving the mixture with ethyl acetate after extraction in a round bottom flask over 72 hours. This effect causes structure 5 to decompose slowly due to additional reaction occurring with ethyl acetate solvent. This experiment increased the percentage yield of structure 5 by 4% (0.01g). Rf 0.25( Tol:EA; 2:1).

In the 1H NMR spectrum (Fig..) additional peaks were seen at 2.086 (s, OCOCH3) and 2.06 (s,H-4, CH3) ppm region suggesting an symmetrical anhydride molecule. These chemical shifts match the predicted values (see table).  This is further supported by a strong C=O peak in IR spectrum at  1736.94 cm-1 region. Therefore, one-hour reaction gave more impurities compared to the two hour reaction.

Method 5

The goal of this method is to reach 88% yield and obtain a high mass. The same method was used and the reaction was left for two hours. Copper sulphate was used as a qualitative observation to confirm the absence of pyridine. Sodium bicarbonate was The product had a yield of 40% and 0.14g. Rf 0.24, 0.23( Tol:EA; 3:1). The 1H NMR spectrum showed the three singlet peaks of acetyl groups deshielded  to 1.970, 1.959 and 1.949 ppm, an unknown impurity has been formed.  Moreover, low yield was witnessed in all previous experiments despite of optimising the methodology. This could be due to moisture being present in both acetic anhydride and pyridine bottles. These bottles were sealed but used by several students for the past one year. Therefore, there were higher chances of these reagents containing moisture.  It’s essential to increase the yield to be able to continue and complete the overall reaction. Acetic anhydride and Pyridine were both refluxed and distilled before the 6th experiment to avoid any moisture being present.

Synthesis of 5-(hydroxymethyl) cyclohex-4-ene-1,2,3- triyl triacetate

The next step, involves reducing the carboxylic group from the CR of  structure 5 to a primary alcohol using proposed method by McCorkindale et al published in 1971. This method associates with reducing the diacetyl-resorcylic in diglyme solvent (20ml) with B2H6 in dry THF (0.5M). The reaction was carried out at 0o C under nitrogen condition for 30 minutes.

However, it has been previously reported in several studies, that digylyme is very  difficult to be removed from the reaction. Hence distilled THF was used instead. This is another type of ether like diglyme and is easily removed by evaporation with a low boiling point (660C) in comparison to diglyme (1620C). Compound 5, was dissolved and stirred for 48 hours with distilled THF (20ml) and borane tetrahydrofuran complex solution (1.0 M in THF). The BH3.THF was added drop by drop cautiously. The mixture was then extracted with chloroform from addition of water and washed with sodium bicarbonate to ensure that the reaction goes into completion. The organic layer was then washed with water to get rid of any salts remaining in the layer. The purified layer was then dried with MgSO4  and concentrated under the vacuum. The product had a yield of  62% and one spot appeared on TLC plate and was stained by KMnO4  dye.

Figure 15. Synthesis of 7

The mechanism of action of the above reaction is summarised in fig 14. There are three steps involved. Firstly, borane which is an electrophile attacks the oxygen in the carboxylic group of compound 5. This forms diacyloxyborane  by removal of hydrogen and reacting with a third addition of borane, producing trialkoxyboroxine (Fig..)  the mixture was then mixed with water and chloroform. The role of water was to quench any unreacted borane. Sodium bicarbonate removed unreacted carboxylic acid group and boric acid from the organic layer. Finally, the mixture was washed with water again to get rid of any salt present in the molecule. The organic layer was then dried in MgSO4 and concentrated under vacuum.

This methodology was particularly chosen. This is because it selectively reduces the carboxylic group without affecting the ester groups in the molecule. Moreover, BH3.THF complex was used instead of borane alone, which exists as a gas. Also its reactivity is   reduced when complexed with THF as analysed compared to BH3. Whereas, other reducing agents such as LiAlH4 and NaBH4 were considered before the reaction but not used due to its limitations. LiAlH4, would reduce the ester groups and the carboxylic group found in structure 5. The second reducing agent, NaBH4 only reduces aldehydes and ketone groups, which are not present in compound 5.

Figure….  Proposed curly arrow mechanism to reduce carboxylic acid group to alcohol with BH3.THF.

Formation of compound 6 was analysed by various of analytical techniques.

 

1H NMR spectrum of compound 6

This spectrum demonstrates synthesis of compound 6. There is a broad peak at 8.05 ppm suggests an O-H bond from the new alcohol group. Another peak appeared at 4.01, representing the methylene group from H-8. Since there is no hydrogen to protonate the carbon the multiplicity of the adjacent carbon is singlet. This peak had a multiplicity of a singlet with 2 proton integrations. The three methyl from acetyl groups (OCOCH3)  were still present in compound 6 as three singlet peaks at 2.036, 2.026 and 2.003 ppm. The proton integration of each peak is 9.  The ring protons H-2 to H-6 are also seen. The alkene group, H-2 at 5.7 ppm as a doublet peak with proton integration of 1. The proton H-3 is at 5.57ppm with a triplet peaks. This is because there is coupling from adjacent protons (H-2 and H-4). Whereas, proton H-4 appeared at 5.1349 ppm as dd due to coupling with H-5. The next proton environment, H-5 is shown at 5.82 ppm with proton integration of one.  In summary all the corresponding peaks were seen successfully.

The coupling between the protons were further analysed by the COSY spectrum (fig). This showed that H-7 coupled with H-6 and they both are coupled with H-5 and H-4.  The proton H-8 is coupled with H-2 and H-3. Also a strong coupling is seen between proton H-2 with H-3.

DEPT135 NMR spectrum

Assignment Results from attempt 6-8 DEPT 13C NMR Results from P. Aguilar Predicted DEPT 13C NMR
C-1 140.852 138.3
C-2 128.47 117.22 128.1
C-3 66.43 66.43 68
C-4 69.122 70.01 73.2
C-5 66.081 67.05 74.5
C-6 30.083 65.16 38.7
C-7 65.486 31.06 138.7
C8- C10 (methyl) 20, 19.35, 19.76 21.02, 20.96, 20.79 21, 21,21
C8- C10 (OCOCH3) 170, 169.443, 169.250 170.1,170.6, 170.7

The above table compares structure C against predicted spectrum. However the predicted NMR does not use reference peak such as CDCl3. Therefore, the recommended peaks maybe slightly out of spectrum. Hence the spectrum is also compared against a previous project study. The spectrum obtained are very similar in comparison to both predicted and by P. Aguilar spectrums. The reference signals which represent methyl in acetyl groups appear at 169.5, 169.4 and 169.3 ppm. The positive signals correspond to C-2 (128.5), C-3 (66.4), C-4 (69.12), C-5 (66.1). Carbon environments of 6 and 7 both gave negative signals at 30.1 and 65.5 ppm, respectively.  However, C-6 signal is odd compared to P. Aguilar but its slightly similar to the predicted spectrum. The three methyl carbon signals are deshielded compared to other two chemical shifts. However the 1H NMR is more sensitive and successfully demonstrates synthesis of reduced analogue of shikimic acid.

Table… compares DEPT 13C NMR spectrum from a previous study and literature against recently synthesised compound 6 (see Fig.. for DEPT 13C NMR spectrum)

Figure.. shows structure 5 in a chair conformation to explain the coupling found in proton H-6.

This finding is further explored and analysed against HSQC spectrum.

HSQC Spectrum

This spectrum is very useful in analysing an unknown molecule. The three acetyl groups at 2.036, 2.026 and 2.003 ppm linked with carbon signals at 20, 19.35 and 19.76 ppm. The proton  H-6, appears as doublet of doublets at 2.7 and 2.66 ppm which correlate with carbon at 30.083 ppm. The proton H-3, has signal at 5.62 and is attached to carbon at 66.43 ppm, whereas proton environment, H-5, at 5.133 and is linked to carbon at 66.081 ppm.

This spectrums confirms the reduction process of forming alcohol group by H-7 at 5.19 is correlated with carbon at 65.486 (see figure..)

Infra-red

The IR spectrum of compound 6 (see fig..) indicated that the carboxylic group being reduced, since there’s no longer a broad strong peak at 3500-2500 cm-1 region. The absorption of hydrogen in alcohol group, O-H is at 2868.55 cm-1, which is in its typical region of the spectrum. Continuing with the analysis, moving to the right of the spectrum C-O stretch is shown at 1227.73 cm-1, confirming the presence of the alcohol group. Whereas, The C=O bending absorption of methyl group appeared at 1370.56 cm-1. While, the ester groups are still present in the compound appeared as a strong peak at 1743.05 cm-1 of the C=O stretching frequency. Finally, another strong peak seen at 1042.48 cm-1 showed C-O bond, which is found in ester group.

GC-MS

This analytical technique also confirms synthesis of alcohol group by forming the following fragments as demonstrated in table ..

Mass to charge ratio (m/z) ratio Ion fragments
242

Molecular formula: C11H15O6

Formula weight

226

Molecular formula: C11H14O5

Formula weight

124

Molecular formula: C7H11O2

Formula weight

Quantitative NMR

Fig. Data analysed from GC-MS spectrum to predict the possible molecular ion fragments of compound 6.

This spectrum further analyses the purity and yield of structure 6. Quantitative NMR was carried out with chloroform as the internal standard after calibration with s-trioxane. To ensure the central of the spectrum was not having a major effect on the determination of concentration. Variation was checked between pure s-trioxane sample against with some water impurities. There was no variation hence only one set of results are presented below. The millimolar calculation of structure 6  and chloroform showed a proportion of 2:1, respectively. Therefore, the product obtained was approximately 33% pure. This is half of expected yield.

Quantifying unknowns using internal chloroform peak as standard

Peak ID From ppm to ppm  value No H in peak Conc./mM RMM Volume / mL Mass /mg
Chloroform Signal 7.251 7.214 202620310.1 1 21.43 119.377 0.800 2.0
 
Compound 1 Peak 1 5.315 5.189 72099037.75 1 7.625387 284 0.800 1.7000
Compound 1 Peak 2 5.141 5.058 75526534.56 1 7.987888 284 0.800 1.8000
Compound 1 Peak 3 4.028 3.911 118187069.9 2 6.24989 284 0.800 1.4000
Compound 1 Peak 4 5.706 5.603 62594535.12 1  6.6201651 284
Compound 1 Peak 5 5.591 5.487 63959765.75 1 6.764556 284 0.800 1.5000
Compound 1 Peak 6 2.693 2.599 73993510.5 2 3.912876 284 0.800 0.9000
Results
Formulas:

Calculation of molar ratio of reduced analogue of shikimic acid

Integral value for CH Chloroform.   62016507.75. AU

Integral value for H of water            14841580.25 AU

Integral value for CH2s of s-trioxane 792605694.3 AU

n(Chloroform) = Integral value for CH Chloroform / 1 x Weight of s-trioxane
Integral value for CH2s of s-trioxane / 6 Molecular mass of s-trioxane

62016507.8 / 1 x

0.021

792605694.3 / 6   90.078
=  

21.89

Moles

However, there were unknown fragments produced along the side of the desired product. There were some starting materials which did not react including structure 5 and THF. Therefore, leave the residue in the vacuum until no change is noticed in the mass. This will ensure THF is evaporated. To avoid the presence of structure 5  in the second step is to carry out proton NMR on the sample to check the progress of the reaction. If there are still some traces of structure 5 then add more of BH3.THF. Since there’s one spot noticed on the TLC plate flash chromatography technique cannot could be used. Therefore, using THF as a possible reagent instead of diglyme requires further investigation to be able to obtain a pure compound 6.

Table.. .

Conclusion and Future work

The aim of this project was to partially synthesise a potential protozoal prodrug with a cyclopropane structure inhibiting the shikimic pathway. This is achieved by mimicking its structure to shikimic acid, which successfully binds onto shikimate kinase enzyme but inhibits the chorismate synthase enzyme.In the experiment shikimic acid was successfully acetylated and reduced.

Due to lack of time, the reaction wasn’t carried out further beyond step two in the synthetic route. The two intermediates produced were analysed by several analytical techniques such as, 1H NMR, 13C-NMR, IR spectroscopy and GC-MS which, showed synthesised correlated signals of acetyl methyl and alcohol groups during the acetylation and reduction process, respectively.

The acetylation process was carried out, which involved dissolving shikimic acid in pyridine and acetic anhydride. The desired product was successfully synthesised, but it was challenging to produce the desired product with minimum impurities, high yield and mass.  Eight attempts were carried out and each attempt was critically analysed to further improve the proposed method by Carbain et al. To further optimise this method six different findings were discovered.

Firstly, the optimum rate of reaction is two hours. The longer the duration of the reaction the higher chance of impurities forming.  Secondly, evaporating the mixture with toluene to remove pyridine was not successful and there were traces of toluene present in the NMR spectrum. Instead pyridine can be effectively removed by mixing the mixture with 1M HCl (20mls) before the addition of ethyl acetate for extraction procedure. A third point to further optimise the removal of pyridine was to add copper sulphate to give a visual indication.  The yield was successfully increased by refluxing and distilling both acetic anhydride and pyridine. The mass was increased by combining three reactions together. Furthermore, the final optimised product obtained, had a yield of 89% (1.16049g). a higher yield was obtained compared to Carbain’s et al study. Therefore, this intermediate was used to synthesis the second intermediate in the synthetic route. However, this product consisted of a chromophore with a pink colour.

The second step in the synthetic route, the reduction process, was successfully carried out. This involved stirring the previously synthesised intermediate with BH3 .THF complex with THF, as an alternative solvent to diglyme. This is due to being easily removed from the mixture. The desired product was formed with yield of .. (mass). However, from all the analytical data, only 1H NMR spectrum had a peak suggesting a carboxylic group. This outcome concluded that the reaction did not go into 100% completion. This could be due to a higher volume of BH3 .THF is required than the proposed Mccarbain et al study.

Additionally, referring to step one, mix all the synthesised 5 from the rest of the five experiments into flash column chromatography using a mobile phase of toluene and ethyl acetate (3:1).  Then carryout data analyse from various analytical techniques to confirm structure 5 before progressing to the second step in the synthetic route.

In the future the second step can be either modified by running the reaction for 30 minutes with distilled THF. Then confirming the reaction with a 1H NMR before the purification method. If the starting materials are still present a few more drops of BH3 .THF should be added.  Alternatively McCarbain et al method can be used. A solution to efficiently extract diglyme could be using a new solvent system such as Et 2O in water. Moreover THF can be  used but the progress of the reaction should be constantly analysed against TLC plate.

Moreover, there are other alternative methods that could be considered in the future to synthesise cyclopropane analogue efficiently. The aim of this step is to form an analogue that would mimic the C=C bond in shikimic acid. The Simmons-Smith reaction is the most recommended method since its cheaper, safer and more environmentally.

An alternative method involves using CPME  to activate the iodomethyl zinc alkoxides which, provides better yield and readily forms a carbenoid species. Furthermore, Furukawa has modified the Simmons-Smith reaction which is most commonly used because of  providing better yields. In addition these proposed conditions avoids the polymerisation of substrates to easily form a zinc carbenoid. This involves using dibromomethane, diethylzinc or diazomethane and zinc iodide to form zinc carbenoid species in a readily rapid reaction. Traditionally, this reaction involves CH2I2 and Zn to form ICH2ZnI.

The next step would involve analysing the desired intermediate to confirm its synthesis with various analytical techniques such as  1H NMR, 13C-NMR, COSY, HSQC, IR and GC-MS.

the final step involves oxidation process using Jones reaction using  CrO3 , H2SO4 , H2O with acetone. This will oxidize the C-7 from an alcohol group to carboxylic acid.  The aim of this reaction is to form an analogue which would mimic the structure of shikimic acid. Flash chromatography technique could be used to purify the compound before collecting analytical data such as NMR, GC-MS, IR  and quantitative NMR.  If synthesis of shikimic acid analogue is confirmed then the next step would involve carryout in-vitro tests.  This is essential to confirm its inhibitory characteristics on protozoal parasite.

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