INSULIN SYNTHESIS

Insulin is a rather small protein, with a molecular weight of about 6000 Daltons. It is composed of two chains held together by disulfide bonds. The figure to the right shows a molecular model of bovine insulin, with the A chain colored blue and the larger B chain green. You can get a better appreciation for the structure of insulin by manipulating such a model yourself. The amino acid sequence is highly conserved among vertebrates, and insulin from one mammal almost certainly is biologically active in another. Even today, many diabetic patients are treated with insulin extracted from pig pancreas.

SYNTHESIS

Insulin is synthesized in significant quantities only in beta cells in the pancreas. The insulin mRNA is translated as a single chain precursor called preproinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin. Insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm.

When the beta cell is appropriately stimulated, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood. C peptide is also secreted into blood, but has no known biological activity.

Methyl Salicylate synthesis

Synthesize Methyl Salicylate

1. Transfer about 60 mL of methanol to the 125 mL Erlenmeyer flask. Add 13.00 g of aspirin to the flask and swirl  until the tablets dissolve. (Aspirin tablets may contain binders that do not dissolve in methanol, which is no cause for concern.)

2. Add about 10.0 mL of concentrated sulfuric acid to the reaction vessel and swirl to mix the solutions. Be careful to add the acid slowly to avoid boiling the solution and causing some of the acidic solution to sputter out of the flask.

3. Clamp the flask to a ring stand and partially immerse it in a hot water bath at about 60°C.

4. Allow the reaction to proceed, stirring the reaction mixture occasionally, for 60 minutes. As the methyl salicylate forms, you’ll notice a distinct wintergreen odor. Keep the reaction mixture at about 60°C, adding water to the bath if necessary to keep its level up. The level of the liquid in the reaction vessel will decrease as methanol evaporates from the flask. Add more methanol as needed to keep the reaction vessel near its original volume.

5. After 60 minutes, stop adding methanol to the flask. Increase the heat slightly to being the liquid in the reaction flask to a gentle boil. Boil the solution long enough to vaporize most of the remaining methanol.

6. When enough methanol has boiled off to reduce the volume in the reaction vessel to about half its original volume, remove the flask from the water bath and set aside to cool.

Part II: Isolate and Purify the Product

The brown liquid in the reaction vessel is a complex solution that contains methanol, crude methyl salicylate, sulfuric acid, unreacted aspirin, and other impurities. Methyl salicylate is freely soluble in methanol, but only very slightly soluble in water. We’ll take advantage of that differential solubility to extract most of the water-soluble impurities from the crude product.

1. Pour the contents of the reaction flask into the sep funnel.

2. Add about 50 mL of ice cold tap water to the sep funnel, cap the funnel, and agitate the contents vigorously for 30 seconds.

3. Allow the contents of the sep funnel to separate into two layers. The aqueous layer contains nearly all of the sulfuric acid and most of any other water-soluble impurities. (Make sure you know which layer is which.) Separate the two layers, and transfer the aqueous layer to the 250 mL beaker.

4. Do a second washing by repeating steps 3 and 4.

SYNTHESIS OF IBUPROFEN

Ibuprofen is available over-he-counter to relieve the pain, stiffness and inflammation that may accompany a number of disorders. it is similar in aspirin the way it works and in the way it can be used, because it acts as an analgesic as well as anti-inflammatory, it is an effective treatment for rheumatoid arthritis, ostheoarthitis, and gout. it also relieves mild to moderate headache, menstrual pains, pain from soft tissue, injuries and following operations.  sometimes Ibuprofen is prescribed along with slower acting drugs in the treatment of Rheumatoid arthritis.

The making of Ibuprofen begins with the compound Isobutyl benzene.

Step1:  a Friedel-Crafts acylation that uses the catalyst aluminum-chloride that generates aluminum trichloride hydrate as waste by-product.

Step 2: a Darzens reaction with ethyl-chloroacetate that results in an epoxy ester compound.

Step 3: involves decarboxylation and hydrolyzation forming an aldehyde.

Step 4: a reaction with hydroxylamine giving anoxime.

Step 5: the oxime is converted to a nitrile.

Step 6: hydrolyzation of the nitrile resulting in the final product 2,4-isobutylphenyl-propanoic acid.

This process has a 40% atom economy which translates into 60% waste products. This means that if 30 million pounds of Ibuprofen is produced each year, then more than 35 million pounds of waste is generated.

The green synthesis of Ibuprofen also begins with the compound isobutyl benzene

Step 1: a Friedel-Crafts acylation using hydrogen fluoride as the catalyst that can be recovered and reused.

Step 2: involves hydrogenation with Raney nickel, which is recovered and reused, to produce an alcohol.

Step 3: the alcohol undergoes carbonylation with the catalyst, palladium, also recovered and reused, to produce Ibuprofen or 2,4-isobutyl-phenylpropanoic acid.

This process has a 99% atom economy, includes the recovered acetic acid that was generated in Step 1. This means that only 1% is waste by-products which Translated to less than 500,000 pounds of waste for the production of 30 million pounds of Ibuprofen.

PARACETAMOL SYNTHESIS

Paracetamol also known as Acetaminophen has been known since the early of 1900s, it has only been widely used as an analgesic only since the 1950s. one of the group of drugs known as the non-narcotic analgesics, it is kept in the home to relieve occasional bouts of mild pain and to reduce fever.  the brand names of Paracetamol are Calpol, Disprol, Hedex, and Panadol. Paracetamol are used in the following combined medications Anadin extra, Migraleve, Pameton, Solpadeine, Tylex, and Veganin.

SYNTHESIS OF PARACETAMOL:

Preparation of acetaminophen involves treating an amine with an acid anhydride

to form an amide. In this case, p-aminophenol, the amine, is treated with aceticanhydride to form acetaminophen (p-acetamidophenol), the amide.

The crude solid acetaminophen contains dark impurities carried along the p-aminophenol starting material.

These impurities, which are dyes of unknown structure, are formed from oxidation of the starting phenol.
While the amount of the dye impurity is small, it is intense enough to impart color to the crude acetaminophen. Most of the colored impurity is destroyed by heating the crude product with sodium dithionite (sodium hydrosulfi Na2s2O3) The dithionite reduces double bonds in the colored dye to produce colorless substances. The decolorized acetaminophen is collected on a  Hirsch funnel. It is further purified by a microscale crystallization technique utilizing a Craig tube.

Procedure

Reaction Mixture

Weigh about 0.150 g of p-aminophenol (MW = 109.1) and place this in a 5-mL conical vial. Using an automatic pipet (or a dispensing pump or a graduated pipet), add 0.450 mL of water and 0.165 mL of aceticanhydride (MW= 102.1, d = 1.08 g/mL). Place a spin vane in the conical vial and attach an air condenser.

Heating

 

Heat the reaction mixture with an aluminum block or sand bath at about 115°C and stir gently. If you are using a sand bath, the conical vial should be partially buried in the sand so that the vial is nearly at the bottom of the sand bath. After the solid has dissolved (it may dissolve, precipitate, and re-dissolve), heat the mixture for an additional 10 minutes to complete the reaction.

Isolation of Crude Acetaminophen

Remove the vial from the heat and allow it to cool. When the vial has cooled to the touch, detach the air condenser, and remove the spin vane with clean forceps or a magnet. Rinse the spin vane with two or three drops of warm water, allowing the water to drop into the conical vial. Place the conical vial in a small beaker and allow it to cool to room temperature.

If crystallization has not occurred,scratch the inside of the vial with a glass stirring rod to initiate crystallization. Cool the mixture thoroughly in an ice bath for 15-20 minutes and collect the crystals by vacuum filtration on Hirsch funnel. Rinse the vial with about 0.5 mL of ice water and transfer this mixture to the Hirsch funnel. Wash the crystals on the funnel with two additional 0.5-mL portions of ice water.

Dry the crystals for 5-10 minutes by allowing air to be drawn through them while they remain on the Hirsch funnel. Transfer the product to a watch glass or clay plate and allow the crystals to dry in air. It may take several hours for the crystals to dry completely, but you may go on to the next step before they are totally dry. Weigh the crude product and set aside a small sample for a melting point determination and a color comparison after the next step. 

Calculate the percentage yield of crude acetaminophen (MW = 151.2). 

Record the appearance of the crystals in your notebook. 

Decolorization of Crude Acetaminophen

Dissolve 0.2 g of sodium dithionite (sodium hydrosulfite) in 1.5 mL of water in a 5-mL conical vial. Add your crude acetaminophen to the vial. 

Heat the mixture at about 100°C for 15 minutes, with occasional stirring with a micro-spatula. Some of the acetaminophen will dissolve during the decolorization process:

* Cool the mixture thoroughly in an ice bath for about 10 minutes to reprecipitate the decolorized acetaminophen (scratch the inside of the vial, if necessary, to induce crystallization). 

* Collect the purified material by vacuum filtration on a Hirsch funnel using small portions

(about 0.5 mL total) of ice water to aid the transfer.

* Dry the crystals for 5-10 minutes by allowing air to be drawn through them while they remain on the Hirsch funnel. You may go on to the next step before the material is totally dry.

* Weigh the purified acetaminophen and compare the color of the purified material to that obtained above.

Crystallization of Acetaminophen

Place the purified acetaminophen in a Craig tube. 

Crystallize the material from a solvent mixture composed of 50% water and 50% methanol by volume. The solubility of acetaminophen in this hot (nearly boiling) solvent is about 0.2 glmL. Although you can use this as a rough indication of how much solvent is required to dissolve the solid,you should still use the technique determine how much solvent to add.

Add small portions (several drops) of hot solvent until the solid dissolves. When the solid has dissolved, place the Craig tube in a 10-mL Erlenmeyer flask, insert the inner plug of the Craig tube, and allow the solution to cool. 

When the mixture has cooled to room temperature, place the Craig tube in an ice-water bath for several minutes. If necessary, induce crystallization by gently scratching the inside of the Craig tube with your micro-spatula. Because acetaminophen may crystallize slowly from the solvent, continue to cool the Craig tube in an ice bath for at least 10 minutes.

Place the assembly in a centrifuge (be sure it is balanced by a centrifuge tube filled with water so that both tubes contain the same weight) and turn on the centrifuge for several minutes.

Collect the crystals on a watch glass or piece of smooth paper. Set the crystals aside to air-dry. Very little additional time should be required to complete the drying.

Yield Calculation and Melting Point Determination

Weigh the crystallized acetaminophen (MW = 151.2) and calculate the percentage yield. This calculation should be based on the original amount of p-aminophenol used at the beginning of thisprocedure. 

Determine the melting point of the product. Compare the melting point of the final product with that of the crude acetaminophen. Also compare the colors of the crude,decolorized, and pure

acetaminophen. 

Pure acetaminophen melts at 169.5 -171°C. Place your product in a properly labeled vial.

ASPIRIN SYNTHESIS

 Aspirin is a non-narcotic analgesic that is used over 80 years that relieves pain, reduces fever, and alleviates the symptom of Arthritis. Aspirin's brand names are Caprin, Laboprin, Nu-seals Aspirin, and Solprin.Aspirins are used in the following combined operations such as Antoin, Aspav, Codis, Equagesic, Hypon, Migravess, and Veganin. Aspirin helps to prevent blood clots from forming, and reduces the likelihood of cataracts. it is oresent in numerous combination of medicines for colds, menstrual period pains, headaches, and joints or muscular aches.

The synthesis of Aspirin includes the reactions of Salicylic acid and Acetic Anhydride on a presence of a Catalyst, Phosphoric acid, H3PO4

Once the aspirin is prepared it must be isolated from the reaction solution and purified. The aspirin is insoluble in cold water, and can be isolated by filtering the chilled reaction solution. Purification is necessary to remove any unreacted salicylic acid and acetic anhydride, as well as the acetic acid product and phosphoric acid. Acetic anhydride is caused to decompose by the addition of water once the formation of aspirin is complete

The acetic acid and phosphoric acid are water soluble and can be removed by washing the aspirin with chilled water. Salicylic acid is only slightly soluble in water and is not completely removed in the washing step. Final purification is accomplished by the process of recrystallization. The impure aspirin is dissolved in warm ethanol. The solution is then cooled slowly, and the aspirin crystallizes out of solution leaving the salicylic acid and other impurities behind.

 STEP BY STEP PROCEDURE:

1. Weigh out 3.0 g of salicylic acid and place in a 250 ml Erlenmeyer flask.
2. Measure out 6.0 ml of acetic anhydride and add this to your flask. Be sure to do this in the hood and wear your goggles. Don't let the acetic anhydride contact your skin and don't get the vapors in your eyes.
3. Still in the hood, Carefully add 5 to 10 drops of 85% phosphoric acid, a catalyst, to the flask and swirl to mix everything thoroughly.
   
4. Still in the hood, heat the mixture for about 10 min. in a beaker of warm water (70-80 ^oC).
5. After heating, cautiously add 20 drops of distilled water.
   
6. Next add 20 ml of distilled water and cool in an ice bath. You can do this at your bench. If crystals do not appear, you can scratch the walls of the flask with a stirring rod to induce crystallization.
   
7. Filter the solid aspirin through a piece of pre-weighed filter paper using a Buchner funnel and the aspirator. Wash the crystals with 2-3 ml of chilled water. The liquid is mostly water and can be washed down the sink. Allow the air to be drawn through the solid and filter paper for 15 minutes. Be sure to record the filter paper weight in your notebook.
8. Preweigh a watch glass, and place the filter paper with the product on it and weigh. Obtain the weight of the aspirin by subtracting the weights of the filter paper and the watch glass from the total weight.
9. At this point the aspirin contains traces of water and salicylic acid, and further purification is required. Place a small amount of this impure aspirin in a small labeled beaker, cover with a tissue, and set aside to dry until next week. The amount to be placed in the beaker, enough to measure a melting point, will be indicated by the instructor.
10. transfer the remainder of the impure aspirin to a 100 mL beaker. Add 10 mL of 95% ethanol to the beaker and warm (do not boil) the mixture in a water bath to dissolve the crystals. If the crystals do not all dissolve, add 5 mL more of the ethanol and continue to warm the mixture. When the crystals are all dissolved, add 20 mL of warm water, cover the beaker with a watch glass, and let the solution cool slowly. Crystals of aspirin will form. Complete the recrystallization by cooling in an ice bath.
    
11. Collect the purified aspirin by filtration as before. Dry the crystals by pulling air through them for about 15 minutes. Put the product into a small beaker, cover with a tissue, and set aside to dry further until next week.
12. Measure the melting points of both the impure aspirin, and the recrystallized aspirin.