In the Laboratory
Kinetic Demonstration of Intermolecular General Acid (GA) Catalysis in Thiolysis of 9-Anilinoacridine
W
An Experiment on Enzymology/Physical Organic Chemistry for Advanced Undergraduate/Postgraduate Students Mohammad Niyaz Khan Department of Chemistry, University of Malaya, Pantai Valley, 59100 Kuala Lumpur, Malaysia
Rationale All acids other than hydronium ion (known as specific acid) are called general acids (GA) and all bases other than hydroxide ion (known as specific base) are defined as general bases (GB). If a reaction is sensitive to acid catalysis, it may involve both specific acid and GA catalysis. It is now generally believed that most enzyme-catalyzed reactions involve the occurrence of intramolecular and intermolecular general acid-base (GA-GB) catalysis (1). Since this fact was realized a huge amount of work has been carried out using simpler nonenzymatic reactions with the purpose of developing an understanding of the mechanistic aspects of such catalysis (2). After nearly five decades of active research and numerous publications on GA-GB catalysis, introduction of an experiment on GA for students majoring in enzymology/ physical organic chemistry at advanced undergraduate/postgraduate level is appropriate. A kinetic experiment involving the reaction of 2-mercaptoethanol (2-ME) with 9anilinoacridine (9-ANA) is designed to demonstrate intermolecular GA catalysis. Experimental Procedure 9-Anilinoacridine hydrochloride (9-ANAH+) is prepared from 9-chloroacridine and aniline using the published procedure (3). All solutions involving 2-ME should be freshly prepared in degassed distilled water just before the start of the kinetic runs. Buffer solutions of 2-ME (with 25% free base) and phosphate (with 75% free base at pK2) are prepared by adding the appropriate amounts of NaOH to the standard solutions of 2-ME and H3PO4 or NaH2PO4. The standard stock solution (1.05 mM) of 9-ANAH+ is best prepared in methanol. However, all other standard stock solutions can be prepared in distilled water. 9-ANA absorbs strongly, whereas its thiolytic products absorb comparatively weakly, at 420 nm. Thus, the rate of thiolysis of 9-ANA can be easily studied using usual spectrophotometric techniques. The rate of thiolysis of 9-ANA reveals the occurrence of GA catalysis in this reaction (4). For a typical kinetic run, prepare the reaction mixture (24 mL) containing appropriate amounts of 2-ME buffer or phosphate buffer with 0.04 M 2-ME and potassium chloride (to maintain ionic strength at 1.0 or 1.5 M) and then equilibrate it for about 10 min at 30 °C in a thermostatted water bath. Initiate the reaction by injecting 1.0 mL of 1.05 W Supplementary materials for this article are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/Issues/1998/ May/abs632.html .
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mM 9-ANAH+ solution to the temperature-equilibrated reaction mixture. Start the stopwatch as soon as part of the 9ANAH+ solution is added to the reaction mixture. Quickly shake the reaction mixture and withdraw an aliquot of nearly 2.5 mL from the reaction mixture, transfer it to the 3-mL cuvette, and place it in the thermostatted cell compartment of the spectrophotometer. Record the absorbance reading (Aobs) at 420 nm at time intervals that provide a decrease in Aobs of nearly 0.02 absorbance units. Continue recording Aobs versus time (t) until 10–15 data points are obtained. Record the last data point at time t = t∞ where no detectable change in A obs is observed within about a 30-min time interval. The absorbance at t∞ is represented as A∞. If the spectrophotometer is not equipped with a thermostatted cell holder, withdraw aliquots of about 2.5 mL periodically from the reaction mixture. Transfer each aliquot quickly to the cuvette, and quickly record its absorbance at 420 nm. However, for a fast reaction, a single aliquot may be used to get more than one data point within a period of less than 60 s. Determine the pH of the reaction mixtures at the end of the kinetic runs. Under the present experimental conditions, the thiolysis of 9-ANAH+ obeys the first-order rate law. The observed data (Aobs vs. t) may be used to calculate the pseudo-first-order rate constant (kobs) from eq 1 using the nonlinear least-squares method (5). Aobs = C0εappexp(-kobst) + A∞
(1)
In eq 1 C0 and eapp represent the concentration of 9-ANAH+ at t = 0 and the apparent molar absorptivity, respectively. If a computer and nonlinear least-squares computer programs are not easily accessible, then the slopes of the conventional plots of ln(Aobs – A∞) versus t may be used to calculate kobs. Results and Discussion In order to discover the effects of total 2-ME buffer concentration ([RS]T) of pH 9.03 on the rate of thiolysis of 9ANAH+, five kinetic runs are carried out at [RS]T = 0.2, 0.3, 0.4, 0.5, and 0.6 M. Pseudo first-order rate constants (kobs) follow eq 2 kobs/[RS]T = kn + kb[RS]T
(2)
where [RS]T = [ESH] + [ES᎑] with ESH and ES᎑ represent nonionized and ionized 2-ME, respectively. The nucleophilic second-order (kn) and 2-ME buffer-catalyzed third-order (kb) rate constants for thiolytic cleavage of 9-ANAH+ are calculated from the linear plot of kobs/[RS]T versus [RS] T. The cal-
Journal of Chemical Education • Vol. 75 No. 5 May 1998 • JChemEd.chem.wisc.edu
In the Laboratory
culated values of kn and k b are 9.56 ⫻ 10᎑2 M᎑1min᎑1 and 14.4 ⫻ 10 ᎑2 M ᎑2min–1, respectively. The effect of total phosphate buffer concentration ([Buf ]T) of pH 7.00 on the rate of reaction of 2-ME with 9ANAH+ was determined by carrying out six kinetic runs at a constant [RS]T (= 0.04 M) and at different [Buf ]T (range 0.2 –0.6 M). Pseudo first-order rate constants kobs follow eq 3 kobs = kB[Buf] T
(3)
where [Buf]T = [H2PO4᎑] + [HPO4᎑2] and kB (= kb´[RS]T) is the phosphate buffer-catalyzed apparent second-order rate constant. The value of kB (= 0.380 M᎑1min᎑1) was obtained from the slope of the plot of kobs versus [Buf]T. The relationship: kB = kb´[RS]T yielded kb´ as 9.50 M᎑2min᎑1 because [RS]T = 0.04 M. It is interesting to note that phosphate buffer-catalyzed third-order rate constant kb´ is nearly 60 times larger than the 2-ME buffer-catalyzed third-order rate constant kb. These observations demonstrate that the buffer catalysis involves GA catalysis because the pKa of HOCH 2CH2SH (= 9.45) is larger than pKa of H2PO4᎑ (= 6.42). Equipment The following equipment is needed for these experiments: water aspirator or sonicator, pH meter, stopwatch, 3mL cuvette with stopper, UV-vis spectrophotometer, 100-mL conical flasks with stoppers, graduated pipets, and volumetric flasks.
Chemicals The following chemicals are needed for these experiments: 9-Chloroacridine, aniline, methanol, 9-anilinoacridine hydrochloride, HOCH2CH2SH, H3PO4 or NaH2PO4, KCl or NaCl, KOH or NaOH, HCl Supplementary Material The complete description of this experiment along with supplementary materials are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/Issues/1998/May/ abs632.html. Literature Cited 1. Jencks, W. P. Catalysis in Chemistry and Enzymology; McGrawHill: New York, 1969; Fersht, A. R. Enzyme Structure and Mechanism; Freeman: New York 1977; Jencks W. P. Adv. Enzymol. 1975, 43, 219; Kirby, A. J. Adv. Phys. Org. Chem. 1980, 17, 183. 2. Jencks, W. P. Acc. Chem. Res. 1976, 9, 425; 1980, 13, 161; Chem. Soc. Rev. 1981, 10, 345; Chem. Rev. 1985, 85, 511; Bruice, T. C.; Donzel, A.; Huffman, R. W.; Butler, A. R. J. Am. Chem. Soc. 1967, 89, 2106; Guthrie, J. P. J. Am. Chem. Soc. 1980, 102, 5286; Kershner, L. D.; Schowen, R. L. J. Am. Chem. Soc. 1971, 93, 2014. 3. Denny, W. A.; Atwell, G. J.; Cain, B. F. J. Med. Chem. 1978, 21, 5; Atwell, G. J.; Cain, B. F.; Seelye, R. N. J. Med. Chem. 1972, 15, 611. 4. Khan M. N.; Kuliya-Umar, A. F. Bioorg. Med. Chem. 1995, 3, 881. 5. Lajis, N. H.; Khan, M. N. Pertanika 1991, 14, 193.
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