The Amide Coupling (Day 1)

In this portion of the organic laboratory, you will be using EDCI to couple your chosen carboxylic acids to the chosen protected amino acids in order to synthesized a protected form of your designed compounds. After the amide coupling is complete, the next step will be the amide workup series of washings.

Preparing for lab

The first thing we need to do is prepare your notebook (and you) for carrying out these reactions. This starts by building a reaction scheme and reaction table in your notebook. Use the following template to build your reaction scheme and table. Remember to replace the R groups with the appropriate structures in your scheme based upon your groups choices from the BOMM design day. Choose three of the six and divide them between your group members to that each person constructs a table in their notebook.

Figure 1. Reaction Scheme and Table Template
Figure 1. Reaction Scheme and Table Template

Complete the table by looking up the names and molecular weights for each of your pieces using the Acid Structures PDF and the Protected Amino Acids PDF. Then, multiply the mmol amount in the table by the molecular weight for each line, which gives you the amount of that compound needed to be weighed out in mg. If your amino acid is an HCl salt (check the Protected Amino Acids PDF), then you will need to add some diisopropylethylamine (DIEA) to your reaction table (129.24 g/mol, 0.742 g/mL, 0.174 mL, 1 mmol, 1 equivalent). The last line of your table will be the product (named by the three letter amino acid code dash number of acid, ex. Tyr-69). To complete this product line, determine the theoretical equivalents, mmol, and theoretical yield by completing the entire row of the table. A completed table is shown below.

Figure 2. An example reaction scheme and table
Figure 2. An example reaction scheme and table

Setting up the reaction

We will begin by weighing out the reactants and placing them into our reaction vessel (a large test tube in this case). We need to use analytical balances in order to get the precision needed for our small quantities. Thus, we will use the balances across the hall in the Bioanalytical Lab.

  1. Mass out your desired amount of your carboxylic acid using weigh paper and the analytical balances, place the solid into your test tube.
  2. Mass out the needed amount of your amino acid using weigh paper and the analytical balances, place the solid into your test tube.
  3. Mass out the needed amount of EDC using weigh paper and the analytical balances, but don’t add to your test tube yet.
  4. Return to your hood and add 2 mL of dichloromethane (DCM, a.k.a. methylene chloride) to your test tube
  5. Add a stirbar to your test tube and clamp the tube above the magnetic stirrer in your hood. Stir the reaction at 300 rpm.
  6. Add the EDC to your test tube while it stirs. The addition of the EDC should help your other solids to dissolve in the DCM.
  7. If your amino acid is a hydrochloride salt, then you will also need to add DIEA at this time as well using a disposable syringe and needle.
  8. Mix until all is dissolved. If it doesn’t dissolve fully, see a TA or Dr. Goode
  9. Place a cap on your test tube and allow the reaction to stir at room temperature overnight.

Cleaning Up

Make sure you dispose of all your weigh paper in the waste bin. Close all lids to the reagents and solvent bottles, and clean up around the balance you used in the Bioanalytical lab.