Azosulfonamides: Preparation and Binding to Carbonic Anhydrase A Bioorganic Chemistry Experiment Mary G. Manalang and Hallie F. Bundyi Mount St. Mary's College, Los Angeles, CA 90049 The study of ligand binding to proteins has become increasinelv i m ~ o r t a n in t a laree number of scientific disciplines, such as medicinal chemistry, enzymology, and immunology. Two articles describing experiments that examine protein-ligand binding have appeared in recent issues of this Journal ( 1 . 2 ) .We wish to report a n alternative experiment employing the enzyme carbbnic anhydrase. As ligands we use certain azosulfonamides, which, like many other sulfonamide inhibitors of carbonic anhydrase, form tightly hound 1:l complexes with the enzymes by coordination with the Zn(I1) a t the active site and through additional interaction with vicinal amino acid residues in the active site cleft (3). T h e azosulfonamides have a n intense a b s o r ~ t i o nin thk visible region of the spectrum that undergoecmarked hv~ochromia on c o m ~ l e xformation with carbonic anhv.. drase. These spectral properties ran be conveniently examined usinr the widelv available Bausch and Lomb Spectronir 20 as wefl as with more advanced spectrophotometers for more sophisticated measurements. The ekperiment that we describe consists of two parts: the synthesis of an azosulfonamide followed by a spectroscopic examination of the azosulfonamide and its complex formation with carbonic anhydrase. We begin the experiment in the second semester of our sophomore-level organic chemistry laboratory with the synthesis of one or more azosulfonamide (one laboratorv . .oeriod) and c o m ~ l e t ethe experiment with the spectroscopic studies during the following lahoratory period.
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azosulfonamide that has been used to prohe the binding site structure in carbonic anhydrase by resonance Raman spectroscopy (8). Prontosil. Dissolve 0.6 g (0.006 mol) m-phenylenediamine in 30 mL 1M sodium acetate, and cool the solution in an ice bath. Prepare a solution of 1.0 g (0.006 mol) sulfanilamide and 2 mL (0.012 mol) 6 M HCI in 10mL water. After cooling the sulfanilamideHCI solution in an ice bath, form the diazonium salt by adding, with stirring, 10 mL of ice-cold 0.7 M NaN02. With thorough and constant stirring3 add the cold diazonium solution dropwise to the cold amine solution. Midway through the addition, and again when the addition is complete, adjust to pH 8 (pH paper) with 5%KOH. Continue stirring for 15miu. Filter the product with suction using hardened filter paper (e.g., Whatman #54), wash it with cold water until the filtrate registers a neutral pH (about 200 mL), and maintain suction until the filter cake appears dry. Using protective gloves," remove the filter cake, and crystallize the product from 50% ethanol-water (about 25 mL for each gram of moist filter cake). Allow the hot, filtered solution t o e d at room temperature for 15 min and then in an ice bath for 30 min. Filter the crystals with suction, and dry over CaClz until the next laboratory period. The yield is about 1g; mp 221-23 "C d. 4-Hydrozyazobenzene-4'-sulfonamide. Prepare a solution of 0.66 g (0.006 mol) of phenol and 0.5 mL 5%KOH in 10mL of water. Cool in an ice bath. Diazatize the sulfanilamide, and couple the diazoniurn salt with the phenol as described for Prontosil. Crystallize the product from 60% methanol-water (about 40 mL for each gram of moist filter cake). Proceed with crystallization as described for Prontosil. Yield: 0.9 g; mp 264-65 OC d. Spectroscopic Studies: Free and Enzyme-Bound Azosulfonamide Reagents
Preparation of Azosulfonamides If it is desired to omit the synthesis, one can employ commerciaUy available Neoprontosi12as the azosulfonamide and proeeed directly to the spectroscopic studies. However, we prefer the experience gained in the individual synthesis of compounds with a specific goal in mind for their use. The synthesis is simple and can be completed through recrystallization in a three-hour laboratory period. It consists of the diazotization of sulfanilamide and coupling of the resultingdiazonium salt toasuitable phenol or amine. We usually present to the student the preparation of 2,4-diaminoazobenzene-4'-sulfonamide (Prontosil), 1, and 4-hydroxyazobenzene-4'-sulfonamide,2.
(1) 0.05 M AMPS05buffer, pH 9.0. (2) 1 X 10-'M bovine carbonicanhydrase(BCA)in 0.05 M AMPS0 buffer, pH 9.0. To prepare this solution, dissolve 300 mg BCA (Sigma Chemical Co., 2,500 Wilbur-Anderson units per milligram protein1 in 100 mL huffer. We label the solution with the molar concentration of protein determined from absorbance at 280 nm using rnra = 54.000 W 1cm-I. M BCA prepared hy diluting the 1 X 10.' M solution of (3) 5 X HCA 1:2 with the AMPS0 huff~r. (4) Each student prepares a 1 X 10-3 M stock solution of the azosulfonamide in ethanol and a working solution of the azwulfonamide (2 X 10V M) by diluting the stock solution 1 mL to 50 mL with the AMPS0 buffer.
' Author to whom conespondence should be addressed.
We use Prontoail because of its interesting history both in the development of antibacterial chemotherapy (6) and as one of the first sulfonamides tested and found to he inhibitors of the enzyme carbonic anhydrase (7). Compound 2 is a more recently studied
Neoprontosil is available from Sigma Chemical Co. under the name Azosulfamide. See refs 4 and 5 for its use in carbonic anhydrase studies. Our students mount the solution of amine or phenol to be coupled in an ice bath on a magnetic stirrer. This is not essential but has produced superior results for use. 'Toxicity data are not available for these azosulfonamides. Using the conservative approach, we avoid skin contact by wearing protective gloves. This also prevents the dyeing of fingers and hands. AMPSO, an acronym for 3-[(l,ldimethyl-2-hydroxyethyllamino]2-hydroxypropanesulfonic acid. is available from Sigma Chemical Co. Volume 66
Number 7
July 1969
609
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