1 Experiment 31 Kinetic Isotope Effects Isotopic substitution is a useful technique for the probing of reaction mechanisms. The change of an isotope may affect the reaction rate in a number of ways, providing clues to the pathway of the reaction. The advantage of isotopic substitution is that this is the least disturbing structural change that can be effected in a molecule. Typically, the isotopic substitution is made with an atom that takes part in the reaction. This will produce a primary isotope effect. If the substitution is made with an atom that does not directly participate in the reaction, a secondary isotope effect exists. Each effect can give different mechanistic information. 1 Primary and secondary alkyl halides undergo elimination reactions under basic conditions. It is generally considered that an E2 mechanism is involved, in which the breakage of the C H bond to the halide group and the departure of the halide leaving group occur simultaneously in the bimolecular transition state. For example; If the protium is replaced by the heavier isotope deuterium, a slower rate of elimination will be observed because the C D bond has lower zero-point energy than the C H bond and a higher activation energy for bond cleavage is therefore required. The change in reaction rate that occurs upon isotope substitution is known as kinetic isotope effect.
2 Chem 463 Organic The elimination reactions of 2-phenethyl bromide and 2-phenethyl-2,2-d 2 bromide under identical conditions of attacking base, temperature and solvent will be examined. A large excess of the base sodium ethoxide will be used so that the reaction will be pseudo first order with respect to the bromide. The kinetics of the reactions will be followed by recording the increase in the UV absorbance of the styrene produced. The rate of production of styrene is given by: dx / kc ( 0 x)[naoet] k( c0 x) dt 1 where x is the concentration of styrene at time = t, k is the second order rate constant, c 0 is the initial concentration of the bromide. When the concentration of the base is significantly greater than the initial concentration of the bromide, there is little change in the concentration of the base as the reaction progresses, and the reaction may be considered as a pseudo first order reaction, with a pseudo first order rate constant k /. The pseudo first order rate constant is related to the second order rate constant by: k / = k[naoet]. Integration and manipulation of equation 1 provides us with equation 2: ln(a A ) kt inf t / 2 which is the standard equation for a pseudo first order reaction. The manipulation recognizes that absorbance is directly related to concentration (Beer s Law), and A t and A inf are the absorbances at time = t and infinite time, respectively. We are now able to determine k / from the absorbance of the styrene produced. If we determine both k / H and k/ D, the isotope effect k/ H /k/ D may now be calculated. The protonated bromide was purchased commercially. The deuterated bromide was prepared via the following route, modified from the method described in reference 2(a).
3 The isotope effect is not limited to the compound under study. Isotopic substitution in the solvent used in the reaction will also give rise to a solvent effect, if the solvent is involved in the reaction. Measurements of these isotope effects can give information about mechanisms such as hydrolysis. The uncatalyzed hydrolysis of acetic anhydride in water is a reaction that may be studied by the kinetic solvent isotope effect. The equation, in protonated water is: A kinetic solvent isotope effect will be observed if any of the following change on going from reactant to transition states: a difference in bulk solvent properties, a difference in solute/solvent interactions, a difference in the zero-point energy of the O H (O D) bond in the reacting solvent, or a difference in the zero-point energy of solute bonds that have exchanged with the solvent. A primary kinetic solvent isotope effect will be observed if a proton (deuteron) is transferred from the solvent in the rate-determining step of a reaction. This is the case here. The proposed transition state for the hydrolysis of acetic anhydride is shown at right. The rate-determining step of the mechanism is the initial attack of water on a carbonyl group of the anhydride. This step is an example of general base assisted nucleophilic attack. The first water molecule acts as a nucleophile, attacking the carbon of the carbonyl group. The second molecule of water acts as a general base, assisting in the removal of a proton from the nucleophilic molecule. In this structure, H a provides a primary kinetic isotope effect, and H b contributes a secondary effect. H c does not contribute an isotope effect. The kinetics of this reaction in D 2 O and H 2 O will be measured by by UV spectroscopy. There is a large excess of water in the reaction, and the pseudo first order rate equation can be used to determine the rate constant of the reaction. These reactions are relatively fast, and the kinetics software package on the Cary UV instrument will be used to record the changing absorbances of acetic anhydride at 228 nm.
4 Chem 463 Organic What to Do? Experimental To complete the basic experiment, the two bromides are obtained as unknowns, and their kinetics are studied. To complete the full report, the deuterated bromide is synthesized from the alcohol, the E2 reaction is studied, and the solvent isotope effect is also studied. Preparation of the Mesylate To a solution of the deuterated 2-phenylethanol (500 mg), triethylamine (500 mg) and N,N-4-dimethylaminopyridine (40 mg) in dichloromethane (10 ml), slowly add methanesulfonyl chloride (1.0 g) with stirring. After the addition is complete, continue the stirring for 1 hour. When the reaction is complete, dilute the reaction mixture with dichloromethane (25 ml), and wash the organic solution with 10% hydrochloric acid (2 x 25 ml), and water (25 ml). Dry (MgSO 4 ), filter and concentrate the organic solution to yield the mesylate. Record the yield, IR and 1 H NMR spectrum of the product. Preparation of the Bromide Prepare a mixture of the mesylate, lithium bromide (2 eq), and acetone (4 ml/mmol mesylate). Reflux the mixture for 4 hours (or longer), and allow it to stand overnight at room temperature. Remove the acetone on the rotary evaporator, and add water (25 ml) to the residue. Extract the aqueous solution with ether (3 x 20 ml). Dry (MgSO 4 ), filter and concentrate the organic solution to yield the crude bromide. Purify the bromide by bulb-to-bulb distillation under aspirator vacuum. Record the yield of the deuterated bromide. For both bromides record the IR, 1 H and 13 C NMR, and submit a small amount of the bromide for analysis by mass spectrometry (we may use the gc-ms). Estimate the % deuteration in the product. Bromides as Unknowns Record the IR, 1 H and 13 C NMR spectra, and obtain the mass spectra of the two bromides provided as unknowns. It is not necessary to positively identify the two compounds before beginning the kinetic study. Kinetics of the E2 Reaction Prepare 250 ml of 10-2 M sodium ethoxide solution by carefully dissolving small pieces of sodium in ethanol. Take care to minimize the exposure of this solution to the atmosphere! Place the stoppered solution in a constant temperature bath at 60 o C and allow to equilibrate. Prepare 10 ml of a solution of 2-phenethyl bromide in ethanol by transferring 12 L (equivalent to 16.2 mg) of the bromide to a volumetric flask, and making up to the mark with ethanol. Transfer 1 ml of this solution to a 100 ml volumetric flask, stopper and allow to equilibrate in the 60 o C constant temperature bath. Repeat for the other bromide, as the kinetics are studied simultaneously. Add the sodium ethoxide solution to the 100 ml volumetric flask containing the bromide and make up to the mark with the ethoxide solution. Thoroughly mix the solution and return it to the constant temperature bath. Note the time of addition (t = 0). At time intervals of 15 minutes remove a small amount of the reaction mixture and record its absorbance (A) at 240 nm against the 10-2 M sodium ethoxide solution as a reference. Record the reference once only at the start of the experiment. The styrene produced in the elimination reaction absorbs strongly at this wavelength
5 whereas the absorbance due to 2-phenethyl bromide is only minimal. Continue to monitor the reaction for 2 3 hours. Keep the reaction mixture in the constant temperature bath and record the absorbance readings after 48 and 96 hours to obtain A inf. Calculate the pseudo first order rate constant k / H (or k/ D ). Solvent Isotope Effect For each kinetic run, pipette 3.0 ml of the supplied potassium chloride solutions (0.02 M in H 2 O or D 2 O, equivalent to 16.9 mg in 10 ml) into a quartz cuvette. To start the reaction, inject 20 L of the standard acetic anhydride solution (2.0 M in acetonitrile, equivalent to 208 mg in 2.0 ml) into the cuvette, gently (and quickly) shake the cuvette to mix the reagents. Place the cuvette into the sample beam of the Cary UV instrument, and record the decay of the absorbance of acetic anhydride at 228 nm. Use the kinetics software on the Cary to compute the rate constant. Record the absorbance for either 30 minutes (H 2 O), or 120 minutes (D 2 O). Repeat the kinetic run for each isotope, and average the rate constants. Report Analyze all spectra recorded, and discuss the differences between protonated and deuterated compounds. Calculate the isotope effect k / H /k/ D for both the E2 reaction and the acetic anhydride hydrolysis. Comment on the results of the experiment, on any assumptions made in the kinetic study, and relate the results to the mechanism of the reaction. References 1. Lowry, T.H.; Richardson, K.S. Mechanism and Theory in Organic Chemistry, 3 nd Ed., Harper and Row, 1987 pp 232 244. Carey, F.A.; Sundberg, R.J. Advanced Organic Chemistry Part A, 2 nd Ed., Plenum Press, pp 190 194. 2. (a) Saunders, W.H.; Edison, D.H. J. Am. Chem. Soc. 1960, 82, 138 142. (b) Saunders, W.H.; Williams, R.A. J. Am. Chem. Soc. 1957, 79, 3712 3716. (c) Saunders, W.H.; Bushman, D.G.; Cockerill, A.F J. Am. Chem. Soc. 1968, 90, 1775 1779. 3. The kinetic solvent isotope experiment is adapted from El Seoud, O.A.; Bazito, R.C.; Sumodjo, P.T J. Chem. Ed. 1997, 74, 562 565.