EXPT 146

Synthesis & Bioassay of SulfanilamideFrom K. L. Williamson, R. D. Minard, and K. M. Masters, Macroscale and Microscale Organic Experiments, 5th ed. 2007,

Houghton Mifflin, Boston, p 617; revised 10/18/06

PreLab Exercise

What is the purpose of starting with the amide, acetanilide, in the beginning of the reaction scheme justto convert the amide to the primary amine, sulfanilamide?

Introduction

Paul Ehrlich, the father of immunology and chemotherapy, discovered Salvarsan, an arsenical “magicbullet” (a favorite phrase of his) used to treat syphilis. He hypothesized at the beginning of the 20thcentury that it might be possible to find a dye that would selectively stain (or dye) a bacterial cell andthus destroy it. In 1932 I.G. Farbenindustrie patented a Prontosil a new azo dye, that they put throughroutine testing for chemotherapeutic activity when it was noted that it had a particular affinity for proteinfibers like silk.

NH2N

NH2

N SO2NH2

Prontosil

Prontosil was found to be effective against streptococcal infections in mice, but somewhat surprisingly,it was ineffectual in vitro (outside the living animal). A number of other dyes were tested, but only thosehaving the group were effective. French workers hypothesized that the antibacterial activity had nothingto do with the identity of the compounds as dyes but rather with the reduction of the dyes in the body top-aminobenzenesulfonamide, known commonly as sulfanilamide. On the basis of this hypothesis,sulfanilamide was tested and found to be the active substance.

H2N SO2NHR

Sulfanilamide, R = H

Because sulfanilamide had been synthesized in 1908, its manufacture was not protected by patents, sothe new drug and thousands of its derivatives were rapidly synthesized and tested. When the R groupin sulfanilamide is replaced with a heterocyclic ring system—for example, pyridine, thiazole, diazine,merazine, and so on—the sulfa drug so produced is often faster acting or less toxic than sulfanilamide.Although they have been supplanted for the most part by antibiotics of microbial origin, these drugs stillfind wide application in chemotherapy.

Unlike that of most drugs, the mode of action of the sulfa drugs is now completely understood. Bacteriamust synthesize folic acid for growth. Higher animals, like humans, do not synthesize folic acid andhence must acquire it in their food. Sulfanilamide inhibits the formation of folic acid, stopping the growthof bacteria; and because the synthesis of folic acid does not occur in humans, only bacteria areaffected.

A closer look at these events reveals that bacteria synthesize folic acid using several enzymes,including one called dihydropteroate synthetase, which catalyzes the attachment of p-aminobenzoicacid to a pteridine ring system. When sulfanilamide is present, it competes with the p-aminobenzoicacid (note the structural similarity) for the active site on the enzyme.

HN C

Folic Acid

N

N

N

N

H2N

OH

ON CH H

H2C

H2C

O

OH

O OH

N CO

OHH

H

p-Aminobenzoic acid

Pteridine part Glutamic acid part

p-Aminobenzoic acid part

This activity makes it a competitive inhibitor. Once this site is occupied on the enzyme, folic acidsynthesis ceases and bacterial growth stops. Folic acid can also be synthesized in the laboratory1

The Synthesis and Bioassay of Sulfanilamide

This experiment is the synthesis and bioassay of sulfanilamide, starting with acetanil ide. Acetanilidereacts with chlorosulfonic acid in an electrophilic aromatic substitution. The protecting amide group isremoved from the p-acetamidobenzenesulfonamide by acid hydrolysis. The amide group is more easilyhydrolyzed than the sulfonamide group.

NHO

CH3

AcetanilideMW 135.16mp 114°C

HSO3ClNH

O

CH3

p-Acetaminobenzene-sulfonyl chlorideMW 233.68

NH3

NHO

CH3

SO2Cl SO2NH2

p-Acetaminobenzene-sulfonamideMW 214.25

dil. HCl

NH2

SO2NH2

SulfanilamideMW 172.20,mp 163-164°C

IN THIS EXPERIMENT perfectly dry acetanilide is treated with chlorosulfonic acid, a highly reactivereagent. The hydrogen chloride evolved is trapped; the reaction mixture is added carefully to water; andthe product, p-acetaminobenzenesulfonyl chloride, is isolated by filtration. This solid is added toaqueous ammonia to form p-acetaminobenzenesulfonamide, which is hydrolyzed with hot hydrochloricacid. The resulting solution is neutralized with sodium bicarbonate to give sulfanilamide.

1L. T. Plante, K. L. Williamson, and E. J. Pastore in Methods in Enzymology, eds. D. B. MCCormick and L. D. Wright (NewYork: Academic Press, 1980), 66:533.

CAUTION: Chlorosulfonic acid is a corrosive chemical and reacts violently with water. Withdraw with apipette and pipette. Neutralize any spills and drips immediately. The wearing of gloves and handling inthe hood is required.

The chlorosulfonation of acetanilide in the preparation of sulfanilamide is conducted without solvent in a25-mL Erlenmeyer flask. Fit the Erlenmeyer flask with a septum connected to a short length ofpolyethylene tubing leading into a reaction tube that contains a small piece of damp cotton to traphydrogen chloride vapors (Figure 1). Place 0.25 g of acetanilide in the Erlenmeyer flask. Add 0.625 mLof fresh chlorosulfonic acid (pick up from the stockroom!) a few drops at a time to acetanilide using aPasteur pipette (not a syringe with metal needle). Connect the flask to the gas trap between additions.In 5–10 min the reaction subsides, with only a few small pieces of acetanilide remaining undissolved.

Figure 1. Chlorosulfonation apparatus.

When this point has been reached, heat the mixture on a sand bath for 10 min to complete the reaction,cool the flask in ice, and deliver the oily product by drops with a Pasteur pipette while stirring it into 3.5mL of ice water contained in a 10-mL Erlenmeyer flask in the hood. Should the material in the flasksolidify (it usually does not), add 3.5 g of ice to the reaction flask. Use extreme caution when adding theoil to ice water and when rinsing out any containers that have held chlorosulfonic acid. Rinse the flaskwith cold water and stir the precipitated p-acetaminobenzenesulfonyl chloride for a few minutes until an

even suspension of granular white solid is obtained. Collect and wash the solid with water on a Hirschfunnel.

After pressing and draining the solid, transfer the solid to the rinsed reaction flask, add 0.75 mL ofconcentrated aqueous ammonia solution (ammonium hydroxide) and 0.75 mL of water, and in the hoodheat the mixture over a hot sand bath to just below the boiling point with occasional swirling. Heat themixture in this manner for 5 min. During this treatment, a change can be noted as the sulfonyl chlorideundergoes transformation to a more pasty suspension of the amide. Cool the suspension well in an icebath, collect the p-acetaminobenzenesulfonamide by suction filtration, press the solid on a Hirschfunnel, and allow it to drain thoroughly. Any excess water will unduly dilute the acid used in the nextstep.

Transfer the still-moist amide to a well-drained reaction flask, add 0.25 mL of concentrated hydrochloricacid and 0.5 mL of water, boil the mixture gently until all the solid has dissolved (5–10 min), and thencontinue the heating at the boiling point for an additional 10 min (do not evaporate to dryness). Thesolution, when cooled to room temperature, should deposit no solid amide, but if it is deposited, heatingshould be continued for a further period. The cooled solution of sulfanilamide hydrochloride is shakenwith granulated decolorizing charcoal and filtered by removing the solution with a Pasteur pipette.

Place the solution in a 30-mL beaker and cautiously add an aqueous solution of 0.25 g of sodiumbicarbonate while stirring to neutralize the hydrochloride. After the foam has subsided, test thesuspension with indicator paper; if it is still acidic, add more bicarbonate until the neutral point isreached. Cool thoroughly in ice and collect the granular, white precipitate of sulfanilamide. Weigh thiscrude product. The crude product (mp 161°C–163°C) on crystallization from alcohol or water affordspure sulfanilamide (mp 163°C–164°C) with about 90% recovery. Weigh the recrytalllized sulfanilamideto determine the percent yield and take a melting point. Save 10-15 mg of sulfanilamide for an IRanalysis; see the inside back cover of the lab guide for preparation instructions for analysis. Theremainder of the sample can be used for the bioassay.

Cleaning UpRinse the cotton in the trap with water, add this rinse to the combined aqueous filtrates from allreactions, and neutralize the solution by adding either 3 M hydrochloric acid or sodium carbonate. Flushthe neutral solution down the drain. Any spilled drops of chlorosulfonic acid should be covered withsodium carbonate; the resulting powder should be collected in a beaker, dissolved in water, and thenflushed down the drain.

Bioassay of SulfanilamideBioassay your synthesized sulfanilamide and commercial sulfanilamide with Bacillus cereus to test forbacteriostatic behavior. You will use prepared sterile agar plates and inoculate them with with Bacilluscereus to test for its antibiotic behavior. Try to share an agar plate with a labmate. Each student canuse one half of the plate.

It is very important that the following procedure is observed or contamination from other bacteria willinterfere with the results of the experiment. Whenever you handle the sterile agar petri dishes try tominimize any exposure to random forms of bacteria or dirt in the laboratory. Always wear gloves thathave been sprayed with ethanol when handling the plates and the cultures, because your hands caneasily contaminate the agar. Open the cover of the dish a few inches vertically so that you have justenough room to swab the plate with a known strain of bacteria or insert a testing disk and immediatelycover the agar to reduce the risk of outside contamination. You will use a pair of forceps to handle thetest disk and sterile swabs to streak the plates with the cultures. After preparing your assay you willobserve the growth of bacteria on the plate. If the bacteria do not grow in the area surrounding the disktreated with your essential oil then one or more of the compounds in this isolated mixture is exhibitingantibiotic behavior. See Figure 2 for an example agar plate.

“Working with Biohazards”The following instructions are to be used whenever handling bacterial cultures in the lab.

1. As with any experimental laboratory procedure, safety glasses must be worn at all times!2. Wash your hands with antibacterial soap.3. Put on a new pair of disposable gloves and spray them lightly with ethanol.4. Prepare all bench surfaces for bacterial experiments by wiping them down with either ethanol or

antibacterial wipes or spray. Your TA will inform you of the location of these materials. Allpaper towels used in the preparation of bench spaces can be thrown in the regular garbagecans.

5. Line the clean workspace with a piece of clean bench paper, see TA for location.6. Assemble all glassware and laboratory materials listed in the instructions and place them on the

bench paper in the order in which they will be used.7. The materials that will come in contact with the bacteria must remain sterile until inoculation,

otherwise you will have interfering results from naturally-occurring bacteria. If any supplies arewrapped, do not remove the protective covering until you are ready to use them. You may alsoneed to sterilize some equipment just prior to use and this can easily be accomplished byspraying or dipping these tools in ethanol. All forceps used any bacterial experiments will besterilized by dipping them in ethanol. Remember the ethanol must be allowed to evaporate fromthe surface of the tools, otherwise it will kill the desired bacteria.

8. Whenever you are transferring bacterial cultures it is important to keep any materials that areused in the process contained. Do not lie anything down that has touched the bacteria.Anything that comes in contact with the bacteria must be either sterilized or disposed ofin the biohazard waste. All cotton tipped applicators must be immediately placed in theorange waste containers labeled biohazard. All forceps that come in contact with bacteria mustbe sterilized by dipping them in alcohol.

9. Once the bacterial transfer is complete, the cultures are to be returned to the TA, and the benchpaper is to be folded and placed in the biohazard waste.

10. Whenever you are handling agar plates inoculated with bacterial cultures, you must be wearingdisposable gloves.

11. All used agar plates must be disposed of in the biohazard waste.Whenever you are finished handling the bacterial cultures and any materials that have come in contactwith the cultures dispose of your gloves in the biohazard waste and wash your hands with handsanitizer and then with antibacterial soap.

Procedure for the transfer of sterile cultures:All students must be wearing gloves during this entire procedure.

1. Obtain a prepared, sterile agar plate from a designated refrigerator. Try to coordinate yourbioassay time such that you and another student share a plate.

2. Label the nutrient agar plate with your last name, course, section, and the name of thecompounds(s) that will be placed on the plate. The plate is to be divided into 4 equal quadrants.Each student will have two of these quadrants (labeled), one for their control (commerciallyavailable sulfanilamide) and one for their product. Use the marking pen to delineate thesequadrants and use this area as a guide preparing the plates. Be very careful not to removethe cover of the plate during this process.

3. Next, the bacteria (see stockroom) will be transferred to the plate using the following steps:a. Insert a sterile swab into the culture tube containing the bacteria.b. Remove the swab from the culture tube and then tilt the cover of the agar plate at an

angle to keep exposure to unwanted bacteria to a minimum. Do not lay the top of theplate on the lab table and do not allow anyone to breath come in close contact with theplate.

c. The moist swab is brought in contact with the agar using a rubbing motion. Start at thecenter of the plate and very gently rub back and forth moving toward on edge of theplate. Turn plate 90 degrees and repeat the rubbing of the swab. Turn the plate 135degrees and rub the remaining section of the plate. The plate should now be evenlycoated with the bacterial culture.

d. Replace cover on culture dish.e. Throw the used sterile swab in the biohazard waste container.

4. Next, the synthesized sulfanilamide will be placed in contact with the inoculated culture (seeFigure 2).

a. Add a spatula-tip full of synthesized sulfanilamide product to the plate.5. Next, commercially available sulfanilamide will be placed on the plate (see Figure 2).

a. Add a spatula-tip full of commercially available sulfanilamide to the plate.6. Wrap the edges of the dish with Parafilm.

Place the plate in your locker. The results of the assay can be assessed after 24 hours or longer.

Figure 2. Agar plate (inoculated with B. cereus) containing a sample of synthesized sulfanilamide anda sample of commercially-available sulfanilamide; picture taken before the bioactivity results wereknown.

Cleaning UpPlease refer to the section above, “Working with Biohazards” on clean-up instructions for the bioassaywaste items.

Final ReportBe sure to include a discussion the bioactivity of synthesized sulfanilamide versus that of commercially-available sulfanilamide. Measure the distance of inhibition around the samples.

PostLab Questions

1. What happens when chlorosulfonic acid comes in contact with water?

2. What happens when p-acetaminobenzenesulfonyl chloride is allowed to stand for some time incontact with water?