BOMM Biofilm Inhibitor Synthesis

The procedures for the synthesis of the potential biofilm inhibitors are documented here. The procedures are broken down according to the chemistry being carried out.

Synthetic Overview

The synthesis of the potential biofilm inhibitors used in the BOMM laboratory centers around an amide bond formation between a carboxylic acid and an amino acid. An example compound is shown in Figure 1 below.

Figure 1. Structure depiction of Phe-69.
Figure 1. Structure depiction of Phe-69.

Two separate pieces are combined in an amide coupling reaction: a carboxylic acid and an amine (see Scheme 1).

Scheme 1. General amide coupling reaction.
Scheme 1. General amide coupling reaction.

Amide Couping for Biofilm Inhibitors

In our case, the amine will be an amino acid, which also carries its own carboxylic acid. This will cause issues during the coupling, as there would be two carboxylic acids that could react. In order to avoid this, we will use amino acids that have the carboxylic acid functional group protected (or hidden) as a tert-butyl ester (see Figure 2).

Figure 2. Amino acid compared to a tert-butyl protected amino acid.
Figure 2. Amino acid compared to a tert-butyl protected amino acid.

Thus, an example coupling between 3,5-dibromo-4-methoxycinnamic acid (acid 69) and the protected amino acid phenylalanine (H-Phe-OtBu) is shown in Scheme 2. The coupling reagent is indicated above the reaction arrow and the solvent of the reaction is below the reaction arrow. In our case, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDCI) will be the coupling reagent and methylene chloride (CH2Cl2) will be the solvent.

Scheme 2. Example BOMM biofilm inhibitor amide coupling.
Scheme 2. Example BOMM biofilm inhibitor amide coupling.

After the reaction is complete, the side products of the reaction are removed by washing separately with an acidic aqueous solution and a basic aqueous solution. After evaporation of the organic solvent, the solid protected biofilm inhibitor will remain in the flask.

Removal of the tert-butyl Protecting Group

To complete the synthesis, the carboxylic acid functional group will be deprotected. The tert-butyl ester can be easily cleaved in the mildly acidic but highly polar solvent 2,2,2-trifluoroethanol (HOCH2CF3, TFE). Scheme 3 illustrates the reaction. Typically, stirring in this solvent at room temperature for roughly 48 hours is sufficient to completely deprotect the ester. This process can also be accelerated with the addition of heat to reduce reaction times.

Scheme 3. Example removal of the tert-butyl ester protecting group by TFE.
Scheme 3. Example removal of the tert-butyl ester protecting group by TFE.

A final extraction of the product into basic aqueous solution followed by reacidification and extraction into fresh organic solvent ensures the purity of the final biofilm inhibitor product.