Prontosil Synthesis and Enzyme Binding Studies

Background

Prontosil (2,4-diaminoazobenzene-4’-sulfonamide) is a potential “sulfa drug” used in chemotherapy of especially “cocci infections” (streptococci, gonococci and pneumococci). In literature there are several methods to synthesize this compound and invariably the starting material is sulfanilamide (p-aminobenzenesulfonamide). Sulfanilamide is synthesized in lab from acetanilide treated with chlorosulfonic acid to acetaminobenzenesulfonyl chloride, which upon reacting with ammonia produces acetaminobenzene sulfonamide, and then this compound is hydrolyzed by HCl to sulfanilamide (Williamson, Minard & Masters 2007, p. 617).

Aim

The aim of the first experiment was to synthesize Prontosil by a method in which sulfanilamide was diazotized to 4’ diazochlorobenzenesulfonamide and subsequently azo coupled with m-phenylenediamine to yield Prontosil. The newly synthesized compound was crystallized and recovered in dried state. Physical properties like melting point and percent recovery were determined. Prontosil is a dye, and a substrate and inhibitor of Zn²⁺-containing carbonic anhydrase enzyme responsible for converting CO₂ to HCO₃^– anion (Coleman 1967, pp. 5212-5219).

The aim of the second experiment was to make an assessment of binding property of Prontosil to the binding sites of carbonic anhydrase. The purpose of this titration experiment was to judge at what concentrations of Prontosil and the enzyme, substrate saturation would be achieved. In enzyme substrate kinetics normally hyperbolic curve is obtained while plotting enzyme activity as a function of substrate concentration. At saturation point no further binding of substrate would be possible.

The principle of hypochromia due to binding of colored substrate Prontosil to enzyme binding sites was determined as reduction in absorbance at a wavelength that would be calibrated by taking a wavelength scan (550-350 nm) of the enzyme-free and -bound Prontosil. The hypochromia effect of sulfanilamides and carbonic anhydrase is well documented (Coleman 1968, pp. 4574-4587).

Summary of experimental procedure

Briefly, m-phenylenediamine solution in aqueous sodium acetate trihydrate was prepared in conical flask. Simultaneously, sulfanilamide solution in diluted HCl also was prepared. This solution was used for diazotization by adding sodium nitrite and mixing in another conical flask at chilling temperatures. The m-phenylenediamine and diazotized sulfanilamide solutions were mixed slowly and in installments, and pH adjusted to ~8.0 with sodium bicarbonate.

The crude reaction product, Prontosil, was filtered under vacuum using Buchner funnel, washed in water until water pH reduced to neutrality. The resultant filter cake was first weighed and then dissolved in hot ethanol water mix (1:1 vol/vol). The product was crystallized at room temperature and then in ice bath. The crystallized product was filtered and weighed to constant weight in filter paper. The recovered crystals were then washed in ethanol water mix under suction and dried overnight.

The weight of the dried final processed product was determined with a balance of 0.1 mg accuracy. The melting point range of the final purified Prontosil was determined using a sensitive bomb calorimeter and the values were averaged. For determining the yield the weight of the filter cake immediately after the reaction was taken. Weight of the product after crystallization and following drying of the crystals were also recorded.

In the enzyme binding studies the above purified compound was dissolved at stock concentration equivalent to 10^-3M in 3-[(1,1-dimethyl-2-hydloxyethyl) amino]―2-hydloxyplopanesulphonic acid-NaOH (AMPSO) buffer at pH 9.0. A stock solution of bovine carbonic anhydrase (BCA) was prepared at 10^-4M in the same buffer. These solutions were used for two purposes. To record the visible absorption spectra of free- and enzyme bound Prontosil, diluted working BCA solution (10^-4M) in AMPSO buffer was mixed with an aliquot of Prontosil stock solution in ethanol diluted with AMPSO buffer.

For reference purpose AMPSO buffer, solely, was used. In the third set only Prontosil solution was taken without the enzyme. Using reference tube a wavelength scan of Prontosil and Prontosil-BCA adjunct was taken between wavelength range of 550nm and 350nm. This gave us two wavelengths: one where no difference in readings was seen (410.5nm) and the other in which hypochromia – maximum reduction of absorbance in Prontosil-BCA as against Prontosil – was witnessed (460nm).

Finally, titration was carried out in AMPSO buffer by taking 1 ml of 5 x 10^-5M BCA, to which a serial dilution of Prontosil (0.5 to 4.0 ml from stock) was added. Absorbance at above two wavelengths was recorded for each dilution and the values were plotted against molar Prontosil concentrations. This provided information on substrate saturation (equilibrium) concentration for BCA enzyme.

Results and calculations

Preparation of Prontosil

Calculation: % yield: Theoretical yield of the product (mol) from given equation (refer equations 1-5 in the last page)

Sulfanilamide (1 mol) and m-phenylenediamine (1 mol) gives prontosil (1 mol)

mol of substance used = weight in gram / molecular weight (g mol^-1) — formula 1

Given: 0.60209 g m-phenylenediamine = 0.60209 / 108.14 = 0.0056 mol

1.0065 g sulfanilamide = 1.0065 / 172.2 = 0.0058 mol

1 M HCl = 1 mol L^-1, and final mol of 12 ml of HCl in reaction would be:

[12 (volume of HCl] x 1) / 1000 = 0.012 mol

0.7 M NaNO₂ = 0.7 mol L^-1, and final mol of 10 ml of NaNO₂ would be:

[10 (volume of NaNO₂) x 0.7] /1000 = 0.007 mol

As these mol values are greater than values for sulfanilamide and m-phenylenediamine, they can not be the limiting reactants. The other components are non-essential in reaction.s

Hence theoretical yield of Prontosil should be equal to the minimum mol of the components of the reactants, that is 0.0056 mol

Corresponding weight of Protonsil would be 0.0056 x 291 (molecular weight, see below) = 1.6296 g from formula 1.

Actual yield of the final dried product = 1.5785 g

Theoretical yield of Prontosil from given weights of the reactants = 1.6296

Therefore % yield = 1.5785 x 100 / 1.6296 = 96.86

Melting point = 200^oC (as it started to melt around 196^oC and finished to melt around 204^oC).

Enzyme Binding Studies

Calculation: Molecular weight (g mol^-1) of Prontosil from its empirical formula (C₁₂H₁₃O₂N₅S) is calculated as 291 (C, 12 x 12 = 144; H, 13 x 1 = 13; O, 16 x 2 = 32; N, 14 x 5 = 70; S, 32 x 1 = 32 Total = 291).

Calculations:

• for preparing desired molar solution

Weight of substance (g) = [(mol. Weight (g mol^-1) x desired molarity (M) x volume (ml)] / 1000 —-formula 2

For 10^-3 M solution in 10 ml

Weight (g) = [291 x 10^-3 x 10] /1000 = 0.00291 g or 2.91 mg is to be weighed and dissolved in 10 ml ethanol.

• for preparation of working stock solution

Molarity₁ x Volume₁ = Molarity₂ x Volume₂ (Molarities and volume of main and working stocks) ——formula 3

Here for solution “D” preparation – 10^-3 M x 0.75 ml = Molarity₂ (D) x 50 ml

Hence molarity of “D” = (10^-3 x 0.75) / 50 = 1.5 x 10^-5M

Visible Absorption Spectra of Free and Enzyme-Bound Prontosil

Free Enzyme

Enzyme Bound

Plot of absorbance vs. wavelengths for the free- and enzyme bound Prontosil

Titration of BCA with Prontosil: The working solution “D” (1.5 x 10^-5M) was further diluted to 5 ml in the series to attain the following concentrations of Prontosil

Calculation: use equation 3 for determining molarity of stocks, for e.g.

Stock 1; M₁V₁ = M₂V₂, 1.5 x 10^-5 x 0.5 = M₂ x 5.0

M₂ = (1.5 x 10^-5 x 0.5 / 5.0) = 0.15 x 10^-5 and accordingly calculate other concentrations in stocks 2-8.

Plot between concentration and absorbance at two wavelengths.

Interpretation and discussion of results

Using the chemicals made available the wet weight of the filtered reaction product (crude preparation) was approx. 6.1 g, and a substantial loss in the weight was observed upon crystallization in ethanol water mix, which means about two third of the impurities was removed at this step of purification. It is possible that along with freshly synthesized Prontosil water insoluble compounds at alkaline pH like sulfanilamide (soluble only in dilute acids) and some sodium bicarbonate also co-precipitated. Upon crystallization these impurities were removed. However, apparently though Prontosil is ethanol soluble it seems to be hygroscopic and the crystals retain moisture. The moisture content in crystals is about 20% by weight, which evaporates upon drying thus leaving ca. 26% of the original material.

From the theoretical yield of Prontosil based on mol ratios of the reactants, the yield of experimental product was ca. 96.86%, which from every respect is a very good quantitative yield of the material.

The apparent melting point of purified compound in our experiment was 200^oC which is lower than the published value of Domagk (1935, p. 196) (247-251^oC). However, it should be noted that depending on the physical state the melting point may vary between 168-251^oC. Hence, purity and authenticity of synthesized Prontosil was confirmed.

The visible absorption spectra of free- and enzyme (BCA)-bound Prontosil gave some interesting results. The difference was non-significant at 410.5 nm apparently not discriminating the free-and bound states. At 460 nm maximum difference was attained (bound Prontosil gave 0.032 unit lesser absorbance). This means at 460 nm there was a quenching of the absorbance of Prontosil due to the fact that the compound got bound to the enzyme’s binding site(s).

This phenomenon in reduction of absorbance at specified wavelength of a chromophore is known as hypochromia. It was stated that longer the conjugation of a compound, longer the wavelength of absorption max. Here, enzyme-Prontosil complex has shown a shift of max absorption towards higher wavelength owing to the adduct formation and increase in conjugation (Zinc conjugating with the dye).

From the titration plots between fixed enzyme concentration and variable Prontosil concentrations it is clear that the binding with BCA is a biphasic phenomenon. There was a sharp increase in bound Prontosil in first phase from 0.15-0.3 x 10^-5M which was followed by another steady but slower increasing phase that continued upto 0.9 x 10^-5M. Thereafter, no further absorbance increase was detected even when higher increments of Prontosil were added. Hence 0.9 x 10^-5M may be considered as the equilibrium point for saturation of binding sites of the enzyme. The biphasic nature of enzyme-substrate binding is normally observed once substrate exerts an inhibitory response on its own binding beyond certain concentration.

It should be kept in mind that Prontosil inhibits CO₂ binding to carbonic anhydrase and possibly by same mechanism it also inhibits its own binding. The bound Prontosil also exhibited a similar trend at 410.5 nm, and the titration plot did not intersect the plot recorded at 460 nm, rather it somewhat overlapped the values. It can be concluded that about 0.9 x 10^-5M concentration of Prontosil saturated all the binding sites of BCA at 10^-5M concentration obtained from one-fifth dilution of the working stock (5 x 10^-5M).

Conclusion

The diazotization and azo coupling reactions led to high quality synthesis of Prontosil with substantial yield close to theoretical value. The purified Prontosil was hygroscopic and had melting point somewhat in the range of the commercial product. The BCA binding with purified Prontosil was verified from the absorption difference at two wavelengths and at 450 nm hypochromia effect was seen. BCA-Prontosil titration gave results suggesting enzyme binding saturation of Prontosil at ca. 0.9 x 10^-5M concentration.

Questions

1. Hypochromia means reduction in intensity of absorption of a chromophore or a dye at a wavelength or wavelength band.
2. From the plots between absorbance difference at two wavelengths of free- and enzyme bound Prontosil it can be concluded that at 460 nm there is hypochromia effect of the colored compound. Since the enzyme is not interfering with the absorbance, most likely reduction in absorbance of bound vis-à-vis free Prontosil is attributed to effective binding of the substrate on the binding sites of enzyme. The difference corresponds to the amount of the substrate that bonded to the enzyme. At 410.5 nm this effect could not be seen. In order to prove that BCA-Zn²⁺ is involved, the binding experiments need to be done in presence of a metal chelator like ethylenediaminetetraacetate (EDTA) at 1-5 mM concentration, and the hypochromic effect at 460 nm should abolish.
3. The plot determines how much Prontosil at given concentration range of 0.15-1.2 x 10^-5M remains in enzyme-bound state. As the concentration increased initially there was a steep increase in absorbance due to rapid binding of substrate with the enzyme. This first order reaction was followed by another slower kinetics between 0.3-0.9 x 10^-5M concentrations possibly due to substrate inhibition of its own binding. At 0.9 x 10^-5M concentration substrate saturation was attained with 10^-5M concentration of the enzyme, and no substrate bounded further. At this equilibrium we may conclude that there is 1:1 or equimolar binding of the enzyme and Prontosil (0.9 x 10^-5:10^-5M). Apparently, one mol of enzyme binds to one mol of the substrate.
4. Upon slight heating, denaturation will take place and protein would unfold releasing the associated Zn²⁺. Consequently, the Prontosil binding ability will be partially or completely lost. In such situation absorbance of free and enzyme bound Prontosil would remain same (no hypochromic effect) at 460 nm. Further, in titration experiment equilibrium would not be attained.
5. The azosulfonamides must mimic CO₂, the natural substrate for carbonic anhydrase. The 

   

    group seems to mimic 

   

    and hence this is the group that apparently binds to Zn²⁺ of the enzyme.
6. Sulfanilamide, a primary amine, reacts with sodium nitrite in diluted HCl in diazotization process, and in steps the following reactions take place: (a) reaction of amine and HCl yields amine hydrochloride, (b) NaNO₂ reacts with HCl to produce equimolar nitrous acid, and (c) nitrous acid reacts with amine hydrochloride to give aminediazoniumchloride (4’ diazochlorobenzenesulfonamide) and water.
7. 4’ Diazochlorobenzenesulfonamide and secondary amine like m-phenylenediamine undergo an azo coupling reaction. First –N=N- group couples with NH₂– group of the amine to generate transient –N=N-NH- linkage of azo and amine groups. Here, the resultant compound is 4’ diazobenzenesulfonamide aminophenylamine. This compound undergoes rearrangement to produce the final compound, Prontosil (2,4-diaminoazobenzene-4’-sulfonamide).

References

Coleman, JE 1967, ‘Mechanism of Action of Carbonic Anhydrase: Substrate, Sulfonamide, and Anion Binding’, The Journal of Biological Chemistry, vol. 242, no. 22, pp. 5212-5219.

Coleman, JE 1968, ‘Carbonic Anhydrase-Azosulfonamide Complexes Spectral Properties’, The Journal of Biological Chemistry, vol. 243, no. 17, pp. 4574-4587.

Domagk, G 1935, ‘A contribution to the chemotherapy of bacterial infections’, Deutsche medizinische Wochenschrift, vol. 61, pp 250-253.

Williamson, KL, Minard, RD & Masters, KM 2007, ‘Synthesis & Bioassay of Sulfanilamide’, in, Macroscale and Microscale Organic Experiments, 5th ed, Houghton Mifflin, Boston. Web.