Synthesis of a Potential Enzyme Inhibitor

• Delaram Salehifard

Aim

Synthesis and characterisation of Benzocaine.

Introduction

The Fischer esterification of 4-amino benzoic acid is catalysed by an acid is fully reversible.

Method:

1. 3g of 4-aminobenzoic acid was weighed out and placed into a dry 100cm³ round bottom flask (ensuring no residues are left inside the joint).
2. 20 cm³ of methylated spirits was measured out and added to the 4-aminobenzoic acid in the round bottom flask.
3. 3 cm³ of concentrated Sulfuric acid was measured out and added to the round bottom flask mixture (ensuring no residues are left on the joint), a condenser was fit onto the round bottom flask and the mixture was gently swirled.
4. Using a heating mantle, the mixture was heated and upon boiling; refluxed for 30 minutes.
5. At the end of reflux, the heat source was removed and the mixture was allowed to cool to room temperature.
6. Upon cooling, the mixture was gently stirred using a glass stirring rod and Sodium hydroxide solution (20%) was slowly added to the mixture until a neutral pH was attained.
7. The mixture was allowed to stand for 5 minutes before the contents were poured into a beaker containing approximately 70 cm³ of ice, the reaction vessels was rinsed with distilled water and the washings were transferred into the beaker to reach an approximate volume of 150cm³.
8. The product was filtered using a Buchner funnel (washed with some cold water) ensuring that the moisture is completely sucked out.
9. The product was then transferred onto a watch glass and dried in an oven at a temperature no greater than 60^oC. The dry mass was then noted and the product submitted for analysis.
10. Steps1-10 were repeated using Isopropyl alcohol instead of Methylated spirits and the melting point for the product was attained.

Mechanism of action

Step one portrays the protonation of the carbonyl oxygen on 4-aminobenzoic acid where the Sulphuric acid acts as the H+ donor (the regeneration of this proton would establish the Sulphuric acid as a catalyst). This proton transfer results in a delocalisation of positive charge which gives rise to the presence of the three resonance structures portrayed in “step 1- resonance”. Of these three structures, the middle structure (where the positive charge is localised onto the carbon atom) allows for the esterification to proceed as its partial positive charge allows Nucleophilic attack by the Oxygen atom on Methanol; as seen in step two. Following this nucleophilic attack, a protonation and de-protonation occurs (which has a net effect of proton transfer) thus leading to the formation of a water molecule on the carbon atom which cleaves off in the hydrolysis in step 3. This hydrolysis results in a delocalisation of positive charge which gives rise to the presence of the three resonance structures portrayed in “step 4- resonance”. Of these structures, the middle structure where the positive charge is localised onto the carbon atom allows for a de-protonation to occur as the Hydrogen atom donates its electrons to the positive carbon thus neutralising the carbon and forming a double bond. This de-protonation also results in the regeneration of the Sulphuric acid proton which protonated the reactant in step 1 thus establishing

Results

Week one percentage yield:

Mass of reactant: 3g

Mass of product obtained: 2.38g

RMM reactant: 137

RMM product: 165

Reactant/product moles: 0.022

Theoretical yield= 3.62g

% Yield = (Actual yield / theoretical yield) * 100 = 65.75%

Week two percentage yield:

Mass of reactant: 3g

Mass of product obtained: 2.79g

RMM reactant: 137

RMM product: 179

Reactant/product moles: 0.022

Theoretical yield= 3.92

% Yield = (Actual yield / theoretical yield) * 100 = 71.18%

Melting point:

Experimental product 1MP: 85.9-88.4^oC

Literature product 1MP: 88-90^oC

Experimental product 2 MP: 83.5-84.1^oC

Literature product 2 MP: 84^oC

H NMR

4-amino benzoic acid

Benzocaine

13CNMR

4-amino benzoic acid

Benzocaine

DEPT-135

4-amino benzoic acid

Benzocaine

Analysis

The melting points for both products were average reading from three attempts and are reasonably within the literature range. This can be attributed to accurate measurements, clean utensils (thus avoiding impurities) and sufficient drying.

At roughly 66% and 71% the percentage yields for products one and two respectively are reasonably low. This may be due to a number of problems such as, incomplete transfer of reactant into the reaction vessel, not transferring all of the reaction vessel washings for filtering, incorrect filtering technique where some product was allowed to pass through instead of being retained e.g. filtering too fast or incomplete transfer of the product from the filter paper after filtration.

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With reference to the HNMR tables, I have deduced corresponding H atom based on integration, splitting and chemical shift. The chemical shift of an atom depends on the extent of shielding it has, for example a H atom attached to an Oxygen (e.g. H atom number 6 on 4-amino benzoic acid) has less shielding due to the Oxygen atoms’ electronegativity whereas a H atom attached to a C atom has more shielding as carbon is not electronegative and in the case of H atoms number 2 and 3, they are also surrounded by other atoms which give them some shielding. I found locating H atoms 2 and 3 particularly tricky as their quintet splitting pattern and integration of 3 where very misleading however their chemical shift reaffirmed their identity as it is relatively to the left thus indicating a fair amount of shielding.

With reference to the 13CNMR tables I was able to deduce the corresponding Carbon atoms based on two properties, chemical shift and peak height. The chemical shift (in accordance to the level of shielding/position of the C atoms) allowed me to locate peaks for carbonyl carbons (Carbon number 7 in both reactant and product) and more shielded carbon aand the height/integration of the peaks which corresponds to the number hydrogen atoms attached to the C atom in question.

With reference to the DEPT-135 tables I was able to distinguish the difference between the C atoms in accordance with the different number of H attached to each C atom. This technique portrays CH and CH3 atoms as positively phased and CH2 atoms negatively phased. For atoms with the same phasing, I used the chemical shift ( as with 13 CNMR ) to distinguish between the C atoms in question.

In this esterification, the product was maintained in a pH of 7-8. This was done in order to prevent a nucleophilic attack from hydroxide ions which would hydrolyse the product which and reverse the esterification thus converting the product back into the reactant.

Rf values can be used to deduce the polarity of a molecule, where a low Rf value can indicate a polar molecule. This is based on how the molecule interacts with the mobile and stationary phases. For example; a low Rf value is a result of the molecule interacting with the polar stationary phase/silica and not travelling very far up the plate allowing us to deduce that its polar. Based on this theory and the fact that polarity increases with RMM; a larger molecule would be more polar and thus have a lower Rf. I would therefore predict that Isopropyl 4-Aminobenzoate would have a lower Rf value than Benzocaine due to its larger RMM making it more polar than Benzocaine.

References

UNCP. (2014). CNMR spectroscopy. Available: http://www2.uncp.edu/home/mcclurem/courses/chm550/nmr_lec4.pdf. Last accessed 06/03/2014.Chemspider. (2014).4-Aminobenzoic acid.Available: http://www.chemspider.com/953. Last accessed 06/03/2014.

Chemspider. (2014).benzocaine.Available: http://www.chemspider.com/Chemical-Structure.13854242.html?rid=752b9fda-5ccb-49f3-bf93-47ceb79356b4. Last accessed 06/03/2014.

Jim Clark. (2002).THE MECHANISM FOR THE ACID CATALYSED HYDROLYSIS OF ESTERS.Available: http://www.chemguide.co.uk/physical/catalysis/hydrolyse.html#top. Last accessed 06/03/2014.

Chemspider. (2014).4 aminobenzoic acid.Available: http://www.chemspider.com/953. Last accessed 06/03/2014.

Chemspider. (2014).Isopropyl 4-Aminobenzoate.Available: http://www.chemspider.com/Chemical-Structure.78903.html. Last accessed 07/03/2014.