Pre-practical "MUST DO" to be included in your lab-book: 1. Calculate MW of all compounds listed in the experiments, moles and theoretical yield 2. Reaction mechanism 3. What is the role of triethylamine? 4. What is the function of the ethyl ester and tert-butoxycarbonyl groups during the synthesis? 5. Look up ninhydrin and briefly describe how it works 6. Predict the fragmentation pattern and masses of the dipeptide product EXPERIMENTAL: 1. Weigh out glycine ethyl ester hydrochloride (0.37 g, ## mmol) and dissolve in dichloromethane (15 mL) in a round bottom flask. 2. Add triethylamine (1.5 mL, ### mmol) to this solution. Then add N-(tert-butoxycarbonyl)-L- alanine (0.5 g, #mmol) followed by EDCI (0.5 g, # mmol). Connect a reflux condenser to your flask and heat the mixture to reflux on a heating block for 30 mins. 3. Cool the reaction mixture and add dichloromethane (10 mL). Transfer to a separating funnel. 4. Wash with 2M HCI (20 mL), then NaHCO3 solution (2 x 15 mL) [Caution: will generate allow CO2 in contact with acid, ensure to vent separating funnel], and brine (20 mL), dry (Na2SO4) and filter. 5. Analyse the product mixture by TLC to determine the purity (cyclohexane:ethyl acetate 1:2) you can detect the products on the TLC plate using the ninhydrin stain provided. The product has an R+ of ~0.5. Also run a TLC plate spotted with the starting materials glycine ethyl ester, N-Boc-L-alanine, and the product mixture, stain and compare R values. Describe your observations. 6. Remove the solvent by rotary evaporator. The product is obtained as an oil. Calculate a yield. Experiment 3: SYNTHESIS OF A DIPEPTIDE Learning outcomes: 1. Reflux 2. Track product through extraction in separatory funnel 3. TLC, ninhydrin as staining Background The synthesis of peptides is important for studies of their structure and for biological studies. The technique of solid phase peptide synthesis was developed by Merrifield and is used widely for synthesis of peptides containing up to 60 amino acids. You will carry out a solution phase synthesis of a dipeptide. The key reaction involves formation of the peptide bond by coupling the amino group of glycine to the carboxylic acid group of L-alanine. The reaction involves the addition of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) B to a solution containing the 'protected' alanine A; this leads to in situ formation of a mixed anhydride C which then undergoes reaction with the nucleophile D to give the dipeptide product E. R₁ ཨོ རིཡཱསུ ΝΗ с N-R OH R N=C=N-R₁ B H₂N. D E