Background and Theory of the Experiment
Understanding the production of peptides—the building blocks of proteins—by joining amino acids with peptide bonds is the goal of peptide synthesis, a basic area of organic chemistry. Having both an amine (-NH2) and a carboxylic acid (-COOH) functional group on the same carbon atom, amino acids are organic compounds. Different amino acids are produced depending on the precise side chain (R-group) connected to this carbon atom. Polypeptide chains of several amino acids joined by peptide bonds make up proteins essential for biological functions.
Creating methyl N-tert-butoxycarbonyl-L-alanyl-L-phenylalaninate (Ala-Phe-OMe), a diprotected dipeptide, is the primary goal of this work. The reactivity of functional groups creates difficulties for peptide synthesis. Protecting groups temporarily block reactive sites, ensuring selective coupling (Sharma et al., 2022).
One of the relevant amino acids, L-alanine, is shielded from nucleophilic attack during coupling by a tert-butoxycarbonyl (Boc) group on its amino group (Gilbert & Martin, 2011, p. 805). To stop its carboxylic acid group from working as an acylating agent, L-phenylalanine is also protected as a methyl ester. An intermediary called a mixed anhydride is used to create the diprotected dipeptide. The carboxylic acid of N-tert-butoxycarbonyl-L-alanine reacts with an acid chloride (isobutyl chloroformate) to produce this anhydride; N-methylmorpholine’s presence facilitates this coupling process.
Ala-Phe-OMe is formed only in the coupling reaction between N-tert-butoxycarbonyl-L-alanine and methyl L-phenylalaninate hydrochloride. The protective groups prevent undesired reactions between free amino or carboxylic acid groups (Gilbert & Martin, 2011, p. 809). Following the reaction, work-up procedures involving extractions with water and organic solvents are conducted to isolate the diprotected dipeptide, ensuring purification and removal of by-products and reagents.
Procedure
In this experiment, the selective coupling of amino acids prepared a diprotected dipeptide, specifically methyl N-tert-butoxycarbonyl-L-alanyl-L-phenylalaninate (Ala-Phe-OMe). In a 50 mL Erlenmeyer flask, 0.63 g of methyl L-phenylalaninate hydrochloride was added first, followed by 10 mL of dimethylformamide and 0.3 mL of N-methylmorpholine (Gilbert & Martin, 2011, p. 815). The solution was then put in an ice-water bath and designated as Solution A after the flask had been thoroughly mixed.
Then, a 100-mL round-bottom flask with a stir bar received 0.502 g of N-tert-butoxycarbonyl-L-alanine. 0.3 mL of N-methylmorpholine and 10 mL of dimethylformamide were added to this flask, and the mixture was agitated for 5 minutes in an ice-water bath (Gilbert & Martin, 2011, p. 815). The reaction mixture was then constantly agitated in the ice-water bath for 5 to 10 minutes after the addition of 0.4 mL of isobutyl chloroformate was made dropwise (Gilbert & Martin, 2011, p. 815). The reaction was then allowed to continue while cooling in an ice-water bath for 45 minutes after the solution from Solution A was added to the round-bottom flask containing the reaction mixture.
The reaction mixture was diluted with 20 mL of water for work-up, and the resultant liquid was transferred into a separatory funnel (Gilbert & Martin, 2011, p. 815). A 30-mL aliquot of diethyl ether was used to rinse the round-bottom flask, which was transferred to the separatory funnel. When required, the funnel was gently shaken and vented. After separating the layers, the organic layer was washed twice with 25 mL of 1 M HCl, once with 25 mL of a saturated sodium bicarbonate solution, and once with 25 mL of brine.
Anhydrous sodium sulfate was then applied to several spatula tips of the organic layer in a 125-mL Erlenmeyer flask. The liquid was periodically spun for 10 to 15 minutes to accelerate the drying process. If extra anhydrous sodium sulfate was required, it was added. The dried organic solution was carefully decanted into a round-bottom flask and concentrated to about 5 mL to separate and purify the product (Gilbert & Martin, 2011, p. 815).
The solution was then transferred to a 50 mL Erlenmeyer flask and allowed to cool to room temperature. The flask was filled with 15 mL of hexane and rinsed with 0.5-1 mL of diethyl ether. The flask was placed in an ice-water bath to facilitate crystallization and was kept there for 5–10 minutes (Gilbert & Martin, 2011, p. 815). When crystals did not form, techniques from Chapter 3, such as recrystallization and melting point, were used to encourage crystallization. After that, the crystals were separated by vacuum filtering and dried in air.
Results
The experiment’s findings show no crystals developed despite several attempts to induce crystallization using various solvents and techniques. Methyl N-tert-butoxycarbonyl-L-alanyl-L-phenylalaninate (Ala-Phe-OMe), the end product, did not crystallize and remained in an amorphous or non-crystalline state. This lack of crystallization suggests that the final product may have contained significant impurities, hindering the development of a clear crystal structure (Gilbert & Martin, 2011, p. 816).
A frequent problem in organic synthesis is the presence of impurities in the final product. These impurities can come from various causes, including incomplete reactions, side reactions, or inadequate purifying techniques. The solubility and crystallization capacity of the product can both be impacted by impurities.
Multiple attempts at inducing crystallization in this example using various solvent systems, such as hexane and diethyl ether, failed. Crystallization is a popular method of purification used to separate a solid product from a mixture of contaminants. Too many contaminants, however, might prevent the product from crystallizing properly (Gilbert & Martin, 2011, p. 93). Additional purification methods, such as recrystallization using a new solvent system, were required to increase the product’s purity (Sharma et al., 2022). Analytical procedures like thin-layer chromatography (TLC) or nuclear magnetic resonance (NMR) spectroscopy can also be used to determine the purity of the product and locate any impurities.
Table 1: Attempts to Induce Crystallization and Observations
Table 1 shows several attempts to induce crystallization using various solvent systems, with each row representing a separate effort. The consecutive attempt number is displayed in the “Attempt” column. The solvent or solvent mixture used in each attempt is described in the “Solvent System Used” column. Each attempt’s result is detailed in the “Observation” column, which states that no crystals formed and the final product remained amorphous and impure.
Discussion: Post-Lab Exercises
Percent Yield
- The mass of the recrystallized product obtained from the experiment = 0.25 grams.
- Theoretical Yield: The theoretical yield can be calculated based on the amount of starting materials used in the synthesis.
- The initial amount of methyl L-phenylalaninate hydrochloride (Phe-OMe • HCl) used = 0.63 grams.
- The initial amount of N-tert-butoxycarbonyl-L-alanine (Boc-Ala) used = 0.502 grams.
The molecular weights of the compounds are:
Molecular weight of Phe-OMe * HCl = molar mass of Phe + molar mass of OMe + molar mass of HCl.
Molecular weight of Boc-Ala = molar mass of Boc + molar mass of Ala.
Using the molecular weights, the theoretical yield of the dipeptide product (methyl N-tert-butoxycarbonyl-L-alanyl-L-phenylalaninate) is properly (Gilbert & Martin, 2011):
- Molecular weight of Phe-OMe * HCl = 215 g/mol
- Molecular weight of Boc-Ala = 199 g/mol
- Molecular weight of the dipeptide = 398 g/mol.
The theoretical yield of the dipeptide is:
Theoretical Yield = (0.63 g / 215 g/mol) + (0.502 g / 199 g/mol) = 0.00293 mol.
Therefore;
Percent Yield = (Actual Yield / Theoretical Yield) × 100
Percent Yield = (0.25 g / 0.00293 mol) × 100 = 85.4%.
References
Gilbert, M. C., & Martin, S. F. (2011). α-Amino acids and peptides. In J. C. Gilbert & S. F. Martin (Eds.), Experimental Organic Chemistry: A Miniscale and microscale approach (5th ed., pp. 803–816). Brooks/Cole Cengage Learning.
Sharma, A., Kumar, A., de la Torre, B. G., & Albericio, F. (2022). Liquid-phase peptide synthesis (LPPS): A third wave for the preparation of peptides. Chemical Reviews, 122(16), 13516–13546. Web.