Papaine is normally used in separating racemic mixtures with benzoylamino acids. Alternatively the separation can be done using derivatives of amino acids like valine and alanine. The product that is derived after the separation crystallizes with ease. Palarimetry is often used to test whether the resolution has completed successfully. Racemic N-benzoylalanine a synthesis of (R, S)-alanine results from the use of purified benzoyl chloride that is then reacted with aniline and papaine. Upon calming down, aniline becomes coloured due to oxidative processes (Sigel 385). This coloration is eliminated through distillation before use it. To avoid future oxidation aniline is stored in dark bottles. For asymmetric synthesis, the commercial papain should not be highly purified. Commercial papain is highly stable when used together with organic solvents. Papain can be kept at room temperature for a period not exceeding two days at a temperature of 0-5 degrees Celsius; if well stored it can last for a longer period. Papain catalytic activity is depends on thiol group reaction. This is because mild oxidizing agents often deactivate papain by converting the cysteine residues to disulfide (an inactive form). Papain is reactivated when thiol compounds are added to it. The reaction between disulfide compounds and papain regenerate the enzymes necessary for the thiol group reactions. R-cysteine –a thiol activates papain (Antolini 391).
Compared to most proteins, the enzyme papain is smaller. It has a molecular weight of 23, 350. The polypeptide chain of papain has 212 amino acid residues that are joined covalently by amide bonds. It has a three dimensional structure. This was determined by x-ray crystallography.
The report is aimed at addressing additional reactions involved in the resolution of (R, S)-Benzoylamino acids.
The report covers a number of issues regarding the various reactions and reaction mechanisms involved in the resolution of the racemic components of alanine as will be listed in the following discussion.
Resolution of (R, S)-Benzoylamino acids
Materials and Reagents
These include benzoyl chloride, aqueous sodium hydroxide, diethyl ether, methanol, chloroform, and (R, S) alanine.
Proper care should be taken as benzoyl chloride is a lachrymator that irritates and tears the skin. It should only be poured in the hood; Aqueous NaOH corrodes and burns the skin and even very dilute solutions can cause adverse effects to the eye; diethyl ether can easily catch flame therefore no flames should be lit around where it is used. Hot electrical devices should also not be kept around where it is used; methanol should only be used in the hood as it is volatile and flammable; and chloroform is carcinogenic and volatile and should therefore be handled with gloves in the hoods (Antolini 1418).
2.5g crystalline Alanine should be placed in 125 ml Erlenmeyer flask containing a mixture of 22ml distilled water and 3.0 ml 6M NaOH. The mixture should be properly shaken to until a clear solution is obtained. With the gloves on 3.7ml benzoyl chloride should be measured with a measuring cylinder and added to alanine solution in the hood. The graduated cylinder should be rinsed using 3.0ml 6M NaOH and the rinse added to the reaction flask. The flask should then be stoppered using a solid rubber stopper. Erlenmeyer flask should be shaken until layers completely mix. The pH should be monitored (Menabue 1293). On becoming acidic another solution of NaOH should be added for the next 2 minutes followed by shaking. To maintain the pH another solution of NaOH should be added. 15g of ice should be placed in a beaker and 15ml HCl poured on the ice. Reaction solution should be poured into acid ice mixture and stirred. Solid products should be collected by vacuum filtration (Corradi 273).
Papain is an excellent enzyme in separating the racemic components of the benzoylamino acids. At the end of the 7 days, the product precipitates and can easily be observed. The product can be recovered as a solid product through the process of Vacuum filtration. The final product obtained is light tan in color. However, if the experiment proceeds longer than the 7 days, a dark yellow color develop which is as a result of aniline oxidation.
Discussion: Questions and Answers
The purpose of washing the solid product from the enzymatic resolution reaction with 1.0M NaOH solution
The enzyme catalyzed reaction of papain takes place in an acidic environment and the purpose of washing the solid with a neutral solution is to neutralize it since the process of racemization that follows is sensitive to changes in the ionic concentration.
The purpose of controlling the pH of the reaction mixture when (R, S)-benzoylamino acid reacts with aniline in the presence of papain
According to studies that have been carried out, the rate of racemization of free amino acids in aqueous solutions has been shown to be dependent on a number of factors. These factors include; the buffer concentration, ionic strength and the type of buffer among others. For instance, the rate of racemization increases with an increase in the concentration of phosphate ions. Furthermore, it is indicated that ionic strength has no effect on the rate of racemization at pH 7.2 but when the pH increases to about pH 10, the rate increases. Therefore, these changes in the rate of racemization necessitate the control of pH.
The equation for the activation of papain by (S)-(+)-Cysteine hydrochloride
The reaction of the enzyme and the activating compound follows the following reaction mechanism;
R-S.S-R’ + HCl or HCN→R-SH + R’-SCl or R’-SC.
From the equation above, R-S.S-R is the papain-like cysteine and HCl is the (S)-(+)-Cysteine hydrochloride.
Mechanism for the formation of the acyl papain from the Michaelis-Menten complex
Assuming that the reaction for the formation of acyl papain follows the normal acyl addition-elimination reaction pathway, it is possible for an experimenter to determine the formation of the acylated enzyme from the Michaelis-Menten complex. The individual rate constants for the reaction have been shown to be pH dependent.
Experiments that have been carried out indicate that the papain-catalyzed hydrolysis of the benzoylamino acids can be performed over a range of pH values at 35˚C. The mechanism involved in this case is a three-step reaction through a number of intermediates as shown by the following reaction pathway;
E + RCOX ↔ E.RCOX → RCOR + XH →E + RCO2H.
From the above equation, E.RCOX represents the enzyme-substrate complex, ECOR is the acyl papain, RCOX is the substrate, E is the free papain, XH is an intermediate after hydrolysis of the Michaelis-Menten complex and RCO2H is the liberated acyl group.
The report has elaborated on a number of important issues that should be taken into consideration when performing reactions involving resolution of the Benzoylamino acids. The report covered various issues including the factors that affect the reactions involved in the experiment.
As indicated in the discussion above, a number of factors determine the feasibility of the experiment in question; therefore it is important for the experimenter to bear this in mind when performing these kinds of reactions. One such parameter to be considered is the pH value.
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