sl Supporting Information for ‘The Complete Mechanism of an Aldol Condensation Charles L. Perrin,* Kuei-Lin Chang Dept. of Chemistry & Biochemistry, Univ. Calif. San Diego La Jolla, CA 92093-0358 USA Table of Contents Instrumentation 1 Partitioning of Ketol Intermediates 1 Rate Measurements Extraction of Forward Rate Constant 6 Rates of Reaction of Ketol Intermediates 7 Construction of Energy Diagram 8 Supporting References 10 Instrumentation. UV spectra and kinetic data were obtained on a recording UV spectrophotometer. 'H_ NMR spectra were obtained on a 500-MHz NMR spectrometer with CDCI; as solvent. Partitioning of Ketol Intermediates. Each ketol 3 was dissolved in acetonitrile and diluted with HzO to a concentration suitable for UV spectrophotometry, then mixed to 0.01-0.1 M NaOH in a total solution of 10 mL with enough acetonitrile to keep everything dissolved. After 10 half-lives of 3, as measured below, the UV spectrum was taken, Because the UV spectrum of chalcone 4 overlaps the spectra of 1 and 2, it was necessary to measure extinction coefficients at two different wavelengths and solve two simultaneous ‘equations to evaluate the concentrations. Therefore in independent experiments the extinction coefficients of 1, 2, and 4 were measured at both the Amax of chalcone 4, near 312 nm, and the isosbestic wavelength, Aix, Near 250 nm, where €jsb,1 = €isb,2- From the measured absorbances As2 AU ama and Aish the concentrations [1] = [2] and [4] were evaluated by solving eqs $1 I= pathlength. Aisb/l = eis, al] + tis 212] + tsb 4[4] = 2¢isb,tora[l or 2) + eis 414] (sD Amax!l = €max UL) + &max.2[2] + &max.al4]) (S2) Rate Measurements. The rate of base-catalyzed condensation of benzaldehyde 1 plus acetophenone 2 to chalcone 4 was followed by monitoring the absorbance of 4 at its Amax near 312 nm. Concentr ions of benzaldehyde, acetophenone, and NaOH were typically 0.4 to 2 mM, 12 to 40 mM, and 0.1 to 0.4 M, respectively, plus acetonitrile and added NaCl to maintain constant ionic strength. The benzaldehyde was added last and the solution was transferred to an oven-dried and capped 1-,2-, or 10-mm quartz. cuvette. Absorbance values were recorded every 30 seconds until the reaction neared completion, providing 200-300 data points. Because NaOH is a catalyst and because 2 is in excess, neither of their concentrations varies with time. Therefore pseudo-first-order conditions apply, and the third-order kinetics of eq 1 simplifies to eq 2. Although the solution to eq 2 is [ArCHO] = [ArCHO]gexp(-kops!), the spectrophotometer measures the absorbance of product 4, which obeys eq 3. v = d{chalcone//dt = k{ArCHO][ArCOCH3][OH-] a v= d{chaleone}/dt = ~d[ ArCHOVdt = kops[ArCHO] @ A= Ax = (Ax — Andexpkobs!) ) The absorbance data Agps Were fitted to eq 3 by nonlinear least squares. The Solver program in Excel was used to evaluate the three parameters As, Ag, and Kops that minimize Z(Acate — Abs), where Acate is given by eq 3 and where the sum is over all times. Figure SI is an example of the absorbance vs. time curve, under the initial conditions of 1.02 mM PhCHO, 21.6 mM PhCOCH, and 0.100 M NaOH + 0.100 M NaCl in D2O containing 26% acetonitrile. ‘The best fit kops = 1215x104 sl. Tables $1-$10 list reaction conditions, fitting parameters, and ops for all 69 kinetic runs,08 o7 08 05 of 03 02 ° 50 10 150 20280 min Figure $1. Absorbance of 4 (Ar = Ph = Ar’) vs. time, measured at 312 nm in D20 and fit to eq 3 Table SI. Reaction conditions, fitting parameters, and rate constants (s°!, M-2s-1) for reactions of 1 (Ar=Ph) + 2 (Ar'=Ph) in 26% CH3CN + 74% H20. AmM_2mM__OH=M__Ao 108kops —_108%kr 153 42 2 0370 0.296 12.7 102 42 02 0401 0.298 128 ost 42 02 0.388 0277 119 102 21 02 0.219 0.177, 93 102 42 0.1 0.388 0.141 124 1022 2 (Ol 0213) 0.085 89 102 312, 0.1 (0.287 0.107 10.1 102 312 02 0.303 0218 103 od) 312, = 02) = 0240) 0232 110 102 42 01 0372 0.134 115 1024193) 02 0.278 0.248 15 Table S2. Reaction conditions, fitting parameters, and rate constants (s*!, M-?s-!) for reacti 1 (Ar=Ph) + 2 (Ar=Ph) in 26% CH3CN + 74% D20.1mM__2.m} Ao 103kos 102 327 0.303 0.299 Osi 432 0.376 0.376 102 216 0.235 0.264 102 324 0.332 0.166 102 216 0.193 0.121 Osi 216 0.198 0.225 ost 412 0.361 0.339 Osi 15.45 0.166 0.160 153 15.45 0.180 0.169 132 285 0.268 0246 119 132 342 0.405 0.320 14.7 132 248 0.220 0227 114 2.685 24.8, 0.250 0.262 13.2 107 = 248s = 022 | 0228) 0.276 126 107186 0.220.174 0.200 97 107372 0.220.329 0.326 13.2 Table $3. Reaction conditions, fitting parameters, and rate constants (s"!, M-2s-!) for reactions of 1 (Ar=pCIPh) + 2 (Ar'=Ph) in 40% CH3CN + 60% H20. 1mM_24mM__OH-, Ao Aint _10%obs 10% Oiairspe oa) 2 01 0235) 10308 001s as 04175 266 = 0.1. 0472-2318 0.127380. 04175 319 0.1 0.523 2.293 0.146 37.7 (A175 203) | 01 = (0.355. 21083 01121| 430) 04175 309 0.1 0.671 2.366 0.196 41.9 0.2505 39.9 0.1 0.658_1.736 0.205 44.0 ‘Table $4. Reaction conditions, fitting parameters, and rate constants (s"!, M-2sl) for rea 1 (Ar=pCIPh) + 2 (Ar'=Ph) in 40% CH3CN + 60% D20. Ao 10%kops _10%ke 06 26.5 Ol 0.566 1.165 0.167 50.2 06 18.9 Ol eee eee hd 06 «284 = 0.10597 «1175. 0.197 560 (06) 284) = 01 0530. 1761 01173) 491206 06 0.75 ‘Table $5. Reaction conditions, fitting parameters, and rate constants (s"!, M-*s!) for reacti 15.1 22.7 22.7 0.1 0.1 0.1 0.359 0.449 0.584 0.948 1.126 1.334 1 (Ar=pO2NPh) + 2 (Ar'=Ph) in 40% CH3CN + 60% H20. mM. 0.2034 0.2034 0.1356 0.2034 0.2034 24mM. 8.52 5.112 8.52 13.63 1.704 OH-M. 04 0.4 04 04 0.1 A 0.119 0.195 0.181 0344 0.150 Aint 0.551 0.995 0.755 1.167 0.800 0.112 0.139 0.143 10%kobs 0.524 0.361 0.550 0.656 0.182 510 410 48.4 107k 462 454 48.5 39.8 40.2 Table $6. Reaction conditions, fitting parameters, and rate constants (s"!, M-2s-l) for reactions of 1 (Ar=pO2NPh) + 2 (Ar’=Ph) in 40% CH3CN + 60% D2O. 1.mM. 0.2034 0.2034 0.2712 0.2034 2mM__OH-M 852 0 1363 ON ES) (On 5.1201 1.704 0.1 Ao 0.482 0.616 0.827 0.449 0.330 Aint 1.075 1217 1.480 0.999 0.822 10%kos 0.601 0.800 0.856 0.428 0.219 102k 0 48.7 52.0 54.0 48.3 Table 7. Reaction conditions, fitting parameters, and rate constants (s"!, Ms!) for reactions of 1 (Ar=Ph) + 2 (Ar=pCIPh) in 40% CH3CN + 60% H20. Ao Aint 10%Kobs 103 kp 2mM__OH-M 1.mM 0.144 0.144 0.144 0.1584 23.2 0.2187 348 0.2187 30.1 0.2187 30.1 0.2187 18.5 0.464 0561 0.633 0.588 0.941 1.004 1.069 1,083 0.194 0.289 0.274 0.243 28.1 30.6 325 28.9 0.2187 0.144 ‘Table S8. Reaction conditions. 0.384 0841 1 (Ar=Ph) + 2 (Ar=pCIPh) in 40% CH3CN + 60% D20. 1.mM 2.mM Ao Aint 0.169 103kobs 28.8 fitting parameters, and rate constants (s-!, M-2s-!) for reactions of 103 ke OH-M0.144 23.2 0.144 185 0.144 23.2 0.409 0.909 0372 0.801 0.349 0.777 158418. 0.359 860 S6 347 329, 342 31 Table $9. Reaction conditions, fitting parameters, and rate constants (s-!, M-2s-!) for reactions of 1 (Ar=Ph) + 2 (Ar'=pO2NPh) in 40% CH3CN + 60% H30. OH- Ag 1mM__24mM 0.288 3.225 0.288 4.838 0.288 6.45 0.288 5.805 0.2047 0.2047 0.2047 0.2047 0.474 0.735 0.941 0.863 Aint 0.642 0.928 1.142 1.066 10%kobs 0.473 0.503 0.603 0.602 107k 152 146 160 166 036 4.838 0.2047 0.740 0.940 0.571 16.6 Table $10. Reaction conditions, fitting parameters, and rate constants (¢!, M2s"!) for reactions of 1 (Ar=Ph) +2 (A\ OH- Ag }2NPh) in 40% CH3CN + 60% D20. 103kos 10%; 1mM__24mM 0.288 3.225 0.288 4.838 0.288 5.805 0.288 6.45 23.225 0.2046 0.2046 0.2046 0.2046 0.2046 0.483 0.738 0.897 0.998 0.515 0.615 0.845 0.844 0.977 0.613 19.8 24.6 23.3 26.0 198 Extraction of Forward Rate Constant k. Because the condensation reaction does not g0 to completion, it is necessary to extract the forward rate constant k of eq 1 from Kops of eq 2. One method is to plot kobs/[OH-] against [2], as used by Guthrie.’ Because the observed rate is the sum of forward and reverse rates, as in eq $3, the slope of that plot is equal to & and the intercept represents the reverse rate constant ky. However, the slope is overly sensitive to the extremities, at low and high [A‘COCH3]. Kops/[OH-] = KI2] + ky (83) Instead we evaluated the equilibrium constant Ke from the equilibrium concentrations ofST benzaldehyde 1, acetophenone 2, and chalcone 4, as in eq S4, where [2]: is approximated as [2]o because 2 is in large excess over 1, and [I]zc is evaluated as the difference between its initial concentration and the amount that has been converted to 4. Then eq $4, together with eq S5, expressing the equilibrium constant as the ratio of forward and reverse rate constants, can be solved to provide the forward rate constant k, as in eq 4. Tables $1-S10 list forward rate constants k for all 69 kinetic runs. For Ar = Ph = Ar’ the average k of 0.0111+0.0004 Ms"! is, substantially lower than the 0.10 M-s-1 found previously in a aqueous solution but without acetonitrile.! oe le Tl2lx ~ (lo- Ml=)2lo S) Ke = kke (S5) iar Ky = @ OW] T+Ke[2] Because the value of Ke, calculated from eq $4, varies from run to run, an average value was used in eq 4, assuming the same Ke in D0 as in HO. That average was weighted by its standard deviation, calculated by propagation of errors in eq $4, and assuming a constant error in concentration. (The formula recommended for a weighted average uses the variance, but this depends too much on a small number of kinetic runs at low conversion.) Rates of Reaction of Ketol Intermediates. For completeness in the kinetic study Table 3 lists rate constants for base-catalyzed disappearance of ketols 3. By using the partition ratios in Table | each of them can be separated into rate constants for conversion to 4 and reversion to 1 + 2, as also listed in Table 3. Samples of ketol 3 were prepared in H2O with sufficient acetonitrile to guarantee solubility of the products. Reaction was initiated by addition of a small amount of NaOH, sufficient to catalyze decomposition at a convenient rate, and the solution was transferred to a I-, 2, or 10-mm quartz cuvette. Every 15 or 30 seconds the absorbance at the Amax of productS8 chalcone 4 was recorded until the reaction neared completion. Fig. S2 is an example of the absorbance vs. time curve due to reaction of 0.016 M 3 (Ar = pCIPh, Ar’ = Ph) with 0.01 M NaOH. os 05 <4 03 oz Figure $2. Time dependence of 312-nm absorbance due to reaction of 0.016 M 3 (Ar= pCIPh, Ar = Ph) with 0.01 M NaOH, as fit to eq 3. The rate of disappearance of ketol 3 is given by eq S6, and the rate of formation of chalcone 4 is given by eq $7, where R is the partitioning ratio of Table 1. Because the kinetics were followed by monitoring the absorbance of chalcone 4 at its Amax near 312 nm, the data were fitted to eq 3, where Kops = ks[OH-]. d{3\/dt = k3[3][OH"] (6) k v=dlayat= 725 BIOH] (s7) Construction of Energy Diagram. In Fig. | the standard state for all reactants is taken as 1 M. Although rates of enolization vary with solvent,” no correction is made for such variation. The (free) energies of PhCHO (1) and PhCOCH (2) are set at zero. The energy of final product chalcone (4) is evaluated from the equilibrium constant for its formation, averagedso over all kinetic runs. The energy of the transition state for step 5 is evaluated from the rate constant in CH3CN-H0 in Table 2. The energy of the transition state for step 2, relative to that for step 5. evaluated from the partition ratio in Table 1. The energy of ketol 3, relative to the energies of the transition states for steps 2 and 5, is evaluated from the rate constants in Table 3. ‘The energies of enolate PhC(-O-}=CH> and of the transition state of step I leading to it are evaluated from the pK, of 2 and the rate constant for its enolization.' The energies of enolate 5 and of the transition state of step 4 leading to it are evaluated from the C-H pK, of 3, estimated by adjusting the pK, of 2 by the difference between the pKas of hydroxyacetic and acetic acids,’ to take account of the nductive effect of hydroxyl (at the s ime position relative to the -O-), and assuming the same intrinsic barrier as for 2. The O-H pK, of ketol 3 is estimated from the pK, of PhCH2OH,' adjusted by the difference between the pX,s of acetoacetic and acetic acids. The energy of the transition state for step 3 is estimated from the O-H pK, of ketol 3 and an intr ic barrier of 2 keal/mol for O-to-O proton transfer.’ Table S11 lists the energies derived for each species of Scheme 2 and Fig. 1 Table S11. (Free) energies (kcal/mol) of species in Scheme 2 and Fig. | Species _G a 2 #l zh The choice of 1 M standard state for all reactants can make the values in Table $11S10 misleading with regard to the depletion of OH™ catalyst. The steady-state concentrations of anionic species 2~, 3-,and 5 of Scheme 2 would be significant if they were destabilized by only 5.78, 0.31, or 4.53 keal/mol. However, those free energies can be adjusted to 0.1 M OH, 0.02 M PhCOCH3, and 0.001 M PhCHO, as used in these kinetic experiments, by raising the energy of any species containing OH-, PhCOCH3, or PhCHO by RTIn(10), R7In(50), or RTIn(1000), respectively. Then the steady-state concentration of 2~,3-, and § are only 1 x 10-7, 1 x 10°6, and 1 x 109 M, respectively. These are indeed high-energy intermediates, present in negligible concentrations relative to 0.1 M OHC, as claimed, and there is no appreciable depletion of catalyst. Likewise, the steady-state concentration of 3 is only 2 x 105 M, representing no appreciable depletion of 0.02 M PhCOCHS. Supporting References (A) Guthrie, J. P.s Cossar, J.; Cullimore, P. A.; Kamkar, N. M.; Taylor, K. F. Canad. J. Chem 1983, 6/, 2621-2626. (2) Watarai, H.; Suzuki, N. J. Inorg. Nucl. Chem; 1976, 38, 1683-1686. (3) Siani, B.; Angelini, G.; De Maria, P.; Fontana, A. Pierini, M. Org. Biomol. Chem., 2008, 6, 4236-4241 (4) Chiang, Y. Kresge, A. J.; Santaballa, J. A.; Wirz, J. J. Am. Chem. Soc. 1988, 110, 5506- 5510. (5) Jencks, W. P.; Regenstein, J., in Handbook of Biochemistry; Chemical Rubber Co. Cleveland, OH, 2 ed., 1970, pJ-191 (6) Brink, H., Acta Pharm. Suec. 1980, 17, 233-248, via Nakagawa, Y.; Uehara, K.; Mizuno, N. Inorg. Chem. 2005, 44, 9068-9075. (7) Kresge, A. J. Ace. Chem. Res. 1975, 8, 354-360.