Reactivity: Synthesis of the Lewis Adducts of Cu²⁺ with Amino Acids

• To explore the reaction of copper ion with amino acids
• To enhance your skills of observation
• To learn techniques of synthesis and separation

Amino acids polymerize to form proteins (polypeptides), which perform an unbelievable number of crucial functions in living organisms. Although many proteins consist solely of linked amino acids, many others contain metal ions in addition to the protein polymer chain. Such metal ions are attached to the proteins via covalent bonds between the metal and atoms in the polypeptide that have lone electron pairs. Of course, the C=O and NH groups of polypeptides have lone pairs that may be used to form covalent bonds with metals. In addition, some of the amino acids have electron pair donor atoms in their side chains that can bond with metal ions. Examples of these amino acids are methionine (S atom), aspartic acid (-COOH group), threonine (-OH group), and histidine (5-membered ring of 3 C and 2 N atoms; both N atoms have lone pairs). Metal ions that are found attached in this way to protein chains include Fe²⁺ and Fe³⁺, Ni²⁺, Ca²⁺, Mg²⁺, and Cu²⁺. Copper ion is found in the so-called blue copper proteins, which serve to transfer electrons between oxidizing and reducing species in metabolic processes. These proteins get their names from the beautiful blue color imparted to them by the presence of the copper(2+) ion.

In this experiment, you will synthesize, isolate, and characterize one or more complexes of Cu²⁺ with amino acids.

Focus Questions

As you proceed through the experiment, keep the following questions in mind. When you have finished the experiment, provide answers to them.

1. Two procedures for synthesis of copper amino acid complexes are given below. How do the procedures differ?
2. Why is it necessary to add NaOH in the first procedure, but not in the second?
3. What is the likely stoichiometry of your copper complex? Support your answer with as many arguments as you can.
4. Why does acetone cause precipitation of the copper complex?
5. Why is the copper complex not washed with water once it is isolated?

Equipment and Materials

• assorted beakers and flasks
• spatula
• balance
• aspirator trap
• filtration equipment
• amino acids
  • alanine
  • arginine
  • aspartic acid
  • glycine
  • proline
  • threonine
  • methionine
  • histidine
  • tryptophan
• Cu(NO₃)₂.2.5H₂O
• Cu(C₂H₃O₂).H₂O

Safety goggles must be worn at all times in the laboratory. Avoid contact of the reagents with the skin. In the event of skin contact, flush the affected area with copious quantities of cold water.

Record all data and observations in your notebook.

Procedure 1: Reaction of copper ion with an amino acid. Weigh 2 mmole of your amino acid and dissolve in a small volume (less than 5 mL if possible) of water in a small beaker. Calculate the volume of 6 M NaOH required to remove one proton (H⁺) from each mole of your amino acid, then add this volume of 6 M NaOH to the amino acid solution dropwise from a Pasteur pipet. Weigh 0.233 g (1 mmole) of Cu(NO₃)₂.2.5H₂O and dissolve in a small volume of water (2-3 mL if possible). Add the Cu²⁺ solution dropwise to the amino acid solution with swirling. When addition is complete, swirl the beaker for about a minute to allow reaction to come to completion.

For some of the amino acids, the complex will precipitate from solution as the copper nitrate solution is added. For these, the mixture may simply be filtered after swirling to remove the product. The solid may be washed 2 or 3 times with small volumes of acetone, suctioned dry, weighed, and transferred to a 1-dram vial. Calculate percent yield.

For other amino acids, the complex will remain dissolved after addition of the copper nitrate solution is complete. In these cases, it is necessary to find a solvent that will precipitate it. Acetone and ethanol are two possibilities. Place 1-2 mL of acetone in a small test tube and add 2-3 drops of your copper-amino acid solution. If solid forms, acetone is a good choice of precipitating solvent. If no solid forms, repeat the test with ethanol. If solid forms, ethanol is a good precipitating solvent. If neither solvent works, you will have to test other solvents. Once a good precipitating solvent is found, add the colored solution of copper amino acid complex dropwise to a beaker containing about 10 mL of the solvent. Place place the beaker in an ice bath for 5-10 minutes to stimulate crystallization Isolate the product by suction filtration, and wash with a SMALL volume (2-3 mL) of acetone. Suction the product dry, weigh the product, and transfer to a 1-dram vial. Calculate percent yield.

Procedure 2: Reaction of copper ion with an amino acid. Weigh 2 mmole of your amino acid and dissolve in a small volume (less than 5 mL if possible) of water in a small beaker. Weigh 0.199 g (1 mmole) of Cu(C₂H₃O₂)₂.H₂O and dissolve in a small volume of water (2-3 mL if possible, but a little more if necessary). Add the Cu²⁺ solution dropwise to the amino acid solution with swirling. When addition is complete, swirl the beaker for about a minute to allow reaction to come to completion.

For some of the amino acids, the complex will precipitate from solution as the copper acetate solution is added. For these, the mixture may simply be filtered after swirling to remove the product. The solid may be washed 2 or 3 times with small volumes of acetone, suctioned dry, weighed, and transferred to a 1-dram vial. Calculate percent yield.

For other amino acids, the complex will remain dissolved after addition of the copper acetate solution is complete. In these cases, it is necessary to find a solvent that will precipitate it. Acetone and ethanol are two possibilities. Place 1-2 mL of acetone in a small test tube and add 2-3 drops of your copper-amino acid solution. If solid forms, acetone is a good choice of precipitating solvent. If no solid forms, repeat the test with ethanol. If solid forms, ethanol is a good precipitating solvent. If neither solvent works, you will have to test other solvents. Once a good precipitating solvent is found, add the colored solution of copper amino acid complex dropwise to a beaker containing about 10 mL of the solvent. Place place the beaker in an ice bath for 5-10 minutes to stimulate crystallization Isolate the product by suction filtration, and wash with a SMALL volume (2-3 mL) of acetone. Suction the product dry, weigh the product, and transfer to a 1-dram vial. Calculate percent yield.

Clean-up. When you have finished all of your work:

• Clean glassware by recommended procedures, shake off excess water, and return to the Labkit.
• Clean Pasteur pipets, graduated pipets, and vials by recommended procedures, shake off excess water, and place in the drying oven.
• Return all borrowed equipment to the instructor before leaving lab.
• Clean up your work area before leaving lab.

Place all solutions in the appropriately marked waste bottles. After weighing the product, place it in the appropriate waste bottle.

Preparation Questions