Synthesis and Biological Assay of Sulfanilamide Antibacterials Developed by Prof.

John Sowa, Department of Chemistry, Seton Hall University Collaborators: Prof. Carroll Rawn, Department of Biology, Seton Hall University and teaching assistant Jason Kodah. Sulfanilamide was the first compound developed for the treatment of bacterial infections. It was discovered accidentally when biologists first noted that the biological stain known Prontosil was toxic to bacteria. In 1932, German scientist Gerhard Domagk administered Prontosil to a 10-month old boy who was suffering from a staphylococcal infection. The boy made a successful recovery. Further research demonstrated that in vivo Prontosil decomposed to sulfanilamide and it was this compound that acted as an inhibitor of bacterial growth. Soon after sulfanilamide became an extremely important drug for the treatment of bacterial infections. Over the period of 1935 - 1946 it is believed that this drug and related derivatives (known as Sulfa drugs) saved over 250,000 lives. In this laboratory we will investigate pharmaceutical drugs are made, analyzed and tested for biological activity. Specifically you will a) to synthesize sulfanilamide itself, b) determine its identity and purity and c) test the inhibitory affect of this drug on the growth of four different species of bacteria (M. Luteus, E. Coli, B. Subtilis, S. Marcescens). This laboratory also demonstrates the concept of multistep synthesis for all or part of the following reasons: 1) The synthesis of sulfanilamide requires 4 steps. 2) If any step fails you must go back to the start. 3) It involves a protection/deprotection step. Procedure: 1. Synthesis of Sulfanilamide (week 1) The procedure for the synthesis of sulfanilamide is given in the Williamson textbook Expt #46. Follow the schedule and notes below: 1) First lab period: Start with aniline. Perform the protection of aniline, expt #4. This should be a very easy step for to perform. Take a m.p. and IR of the product to prove that the protection step was successful. Measure yield. 2) Second laboratory period: Chlorosulfonation, amination. This is a complex series of steps that must be performed with care. This step requires the use of chlorosulfonic acid which as a strong acid and a lacrymator. Please use this compound only in the hood and use extreme care as it can cause severe burns. If you spill any on you, immediately flush the affected area with copious amounts of water and inform the TA. Remember to always wear your safety goggles. 3) Third laboratory period. Perform the deprotection of acetylsulfanilamide to give sulfonylamide. Take IR and m.p. of product to prove that you have made it.

2. Biological assay (week 2) Cautionary Note: Please make sure that you clean up the surface areas you worked on with a disinfectant to ensure that the bacteria will not spread and/or cause infection. Before leaving the lab, wash your hands thoroughly. Equipment needed: Sterilized Petri dishes (60 mm size) 250 mL Nutrient agar solution in sterilized 500 mL Erlenmeyer flasks - good for 20 plates Bent glass rods (L or T shaped) - flamed for sterilization Sterilized Pasteur pipettes Sterilized tap? Water (boiled) The attached procedure was written by Jennifer Medina, biology major and a sophomore organic student in Spring 1997. This experiment is being performed to check the effectiveness of the sulfanilamides and their derivatives synthesized in the laboratory. Please read the following instructions carefully and note that the organisms being used living bacteria and can be spread if directions are not followed. It is very important to use the appropriate technique when introducing bacteria to the plate and also in the transfer of the bacteria from the bottle to the plate. This technique will be explained to you in class. Each student will receive the following: 9 petri dishes containing nutrient agar. Each student will also need a grease pencil to label the dishes and a bent glass rod. Three different microorganisms will be made available to you on the demonstration desk as well as the sulfanilamide sensitivity discs. It is very important that all students wear gloves at all times during this experiment. Directions: Day 1 See attached diagrams if you need assistance 1. Label all your petri dishes on the lid containing the agar with your initials and the name of the bacteria that will be applied to the plate. One plate will be labeled control for each bacteria. 2. With a Pasteur pipette, introduce 4-5 drops of the bacterial broth to the petri dish. Make sure you cap the bottle immediately afterwards to prevent contamination of the bacteria and the surfaces on which you are working. 3. With a bent glass rod, spread the liquid broth all around the surface of the agar. This will create a "lawn" of bacteria

4. Quickly cover the petri dish and prepare for the next step by obtaining several sensitivity discs that will be soaked in sulfanilamide. Other discs will be soaked in your derivatives. ***New: dissolve the sulfanilamide in dimethylsulfoxide (DMSO) and soak disc in the DMSO solution. Record mg of sulfanilamide/ mL of DMSO used.*** 5. Place the soaked (but not dripping) discs in the center of each corresponding plate and be sure to label the plate's exterior with the contents of the disc. This will not be done for the plates labels "control." 6. Cover the plates and double-check the labeling. * Allow the plates to incubate 24 hours. Directions: Day 2 1. Remove the plates from the incubator and observe any visible growth. If the bacteria does not grow in the area surrounding the disc placed in the center, the bacteria is sensitive to the contents of the disc. This area without growth is called a "zone of inhibition." If there is no such zone, the bacteria is resistant to the contents of the disc. If you have difficulty determining whether there is or is not any growth, compare the plate to your control which should have "confluent" growth, or uninterrupted growth.

Positive Result The area without growth surrounding the disc is the "zone of inhibition" created by the compound. When this zone appears, the compound is effective against the strain of bacteria .

Negative Result Visible confluent growth of bacteria occurs and no "zone of inhibition" is observed. This compound is not effective against this strain of bacteria.

2. In the table below mark + if inhibition is observed and - if inhibition is not observed. M. Luteus control Sulfanilamide 1 Sulfanilamide 2 Sulfanilamide overall Questions for final report: To be presented in a final report and presentation. 1) Explain why the protection step was necessary. Write a chemical equation showing what would happen if the protection step was not used. 2) Write a mechanism for each step of the synthesis. 3) Give a detailed account of how sulfanilamide is thought to work. a) What types of bacteria does it kill. b) Classify the types of bacteria that we are testing. Do the results agree with the data in the literature? E. Coli B. Subtilis S. Marcensens comments