4598
`
`J. Am. Chem. Soc. 1999, 121, 4598-4607
`
`Equilibrium Formation of Anilides from Carboxylic Acids and
`Anilinesin Aqueous Acidic Media
`
`Ahmed M. Aman and R. S. Brown*
`Contribution from the Department of Chemistry, Queen’s UniVersity, Kingston, Ontario, Canada K7L 3N6
`ReceiVed January 11, 1999. ReVised Manuscript ReceiVed March 22, 1999
`
`Abstract: The formations of formanilide, p-methoxyformanilide, p-nitroformanilide, and acetanilide from their
`corresponding carboxylic acids and anilines in aqueous acidic media have been investigated at temperatures
`between 60 and 100 (cid:176)C under a variety of conditions such as pH, D 2O, added phosphate, and added ethanol.
`In each case, the pseudo-first-order rate constants for the establishment of equilibrium (kobs), from both the
`hydrolysis and formation directions, and the conditional equilibrium constant (K¢ ) [anilide]/[aniline]total) were
`determined in excess formate. From K¢, and knowledge of how the p Ka values of RCOOH and anilinium ion
`depend on the various conditions, is derived a corrected equilibrium constant, K¢ eq, defined as [anilide]/
`([aniline][RCOOH]). In the case of formanilide, the K¢ value is found to be invariant with temperature reductions,
`although the K¢ eq value increases. In D2O media, the K¢ value drops slightly, but after correcting for the medium
`induced changes in [aniline] and [RCOOH], the K¢ eq value is the same as in water. In the presence of added
`KH2PO4, the rate of establishment of equilibrium increases but the K¢ and K¢ eq values do not change relative
`to their values without phosphate. Added ethanol is found to increase both the rate of establishment of equilibrium
`and the K¢ equilibrium constants, but reduces K¢ eq. The mechanism of formation of anilides in water under
`acidic conditions is discussed.
`
`Introduction
`
`The great bulk of studies concerning amide hydrolysis are
`conducted under acidic or basic conditions where the reaction
`proceeds essentially to hydrolyzed products.1 Consequently, it
`is generally believed that amide bond formation in aqueous
`solution is unfavorable because the experimental conditions
`under which the reactions are conducted favor the hydrolysis
`process (eq 1). However, from the biochemical perspective, it
`
`has long been known that hydrolytic enzymes are also capable
`of reforming peptides in solution.2,3 The formation of amide
`
`(1) For reviews on this subject, see: (a) Bennett, A. J.; Brown, R. S.
`Physical Organic Chemistry of Acyl Transfer Reactions. In ComprehensiVe
`Biological Catalysis; Sinnott, M. L., Ed.; Academic Press Inc: London,
`1997; Vol. 1, pp 293-326. (b) Brown, R. S. Studies of Amide Hydrolysis:
`The Acid, Base, and Water Reactions. In Biochemical Significance of the
`Amide Linkage; Greenberg, A., Breneman, C., Liebman, J., Eds.; Wiley-
`Interscience: New York, 1999; Chapter 2, in press. (c) Jencks, W. P.
`Catalysis in Chemistry and Enzymology; McGraw-Hill: New York, 1969;
`pp 7-242, 463-554.
`(2) (a) Fruton, J. S. In AdVances in Enzymology; Meister, A , Ed.; J.
`Wiley and Sons: New York, 1982; pp 239-306. (b) Dobry, A ; Fruton, J.
`S.; Sturtevant, J. M. J. Biol. Chem. 1952, 195, 149. Kullmenn, W. J. Biol.
`Chem. 1980, 255, 8234. (c) Carpenter, F. H. J. Am. Chem. Soc. 1960, 82,
`1111. (d) Inovyl, K.; Watanabe. K.; Morihara, K.; Tochino, Y.; Kanaya, T.
`Emura, J.; Sekakibaras, S. J. Am. Chem. Soc. 1979, 101, 751. (e) Esowa,
`Y.; Ohmori, M ; Lchikawa, T.; Kurite, H.; Sato, M.; Mori, K. Bull. Chem.
`Soc. Jpn. 1977, 50, 2762. (f) Westeneys, H.; Borsook, H. Physiol. Re.V.
`1930, 10, 110. (g) Morihara, K ; Oka, T.; Tsuzuki, H. Arch. Biochem.
`Biophys. 1969, 132, 489. (h) Gawron, O.; Glaid, A. J.; Boyle, R. E.;
`Odstrchel, G. Arch. Biochem. Biophys. 1961, 95, 203.
`(3) (a) Homandberg, G. A.; Laskowski, M. Biochemistry 1979, 18, 586.
`(b) Homandberg, G. A.; Mattis, J. A.; Laskowski, M. Biochemistry 1978,
`17, 5220.
`
`bonds in aqueous media in the absence of enzymes is also of
`interest since this is implicated in the origin of life. Attempts4-8
`have been made to form peptides from amines and acids or by
`coupling amino acids under conditions that resemble those of
`primitive earth. In studies simulating so-called prebiotic condi-
`tions in water, linear and cyclic polyphosphates,5 cyanamide,6
`metal ions,7 silica, alumina and clay,8 and more recently iron
`sulfide plus H2S9 have been used for promoting amide bond
`formation from acids and amines.
`Reformation of lactams from their amino acid hydrolysis
`products is well-documented and,
`in many cases,
`is very
`favorable due to the intramolecularity of the process.10 Studies
`by Fersht and Requena,11 Morawetz and Otaki,12 Guthrie,13 and
`
`(4) (a) Oie, T.; Loew, G. H.; Burt, S. K.; Binkley, J. S.; MacElroy, R.
`D. J. Am. Chem. Soc. 1982, 104, 6169. (b) Oie, T.; Loew, G. H.; Burt, S.
`K.; Binkley, J. S.; MacElroy, R. D. J. Am. Chem. Soc. 1983, 105, 2221.
`(5) (a) Chung, N. M.; Lohrmann, R.; Orel, E.; Rabinowiz, J. Tetrahedron
`1971, 27, 1210. (b) Yamanaka, J.; Inomata, K.; Yamagata, Y. Origins Life
`1988, 18, 165. (c) Rabinowitz, J.; Flores, J.; Krebsbach, R.; Rogers, G.
`Nature 1969, 224, 795. (d) Rabinowitz, J.; Hampai, A. J. Mol. EVol. 1985,
`21, 199.
`(6) (a) Steinman, G. D.; Kenyon, D. H.; Calvin, M. Biochem. Biophys
`Acta 1966, 124, 339. (b) Ponnamperuma, C.; Peterson, E. Science 1965,
`147, 1572. (c) Steinman, G.; Kenyon, D. H ; Calvin, M. Nature 1965, 206,
`707.
`(7) Rishpon, J.; O’Hara, P.; Lahav, N.; Lawless, J. G. J. Mol. EVol. 1982,
`18, 179.
`(8) (a) Bujdak, J.; Rode, B. M. J. Mol. EVol. 1997, 45, 457. (b)
`Schwendinger, G. M.; Rode, B. M. J. Mol. EVol. 1992, 22, 349.
`(9) Keller, M.; Blochl, E.; Wachtershauser, G.; Stetter, K. O. Nature
`1994, 368, 836.
`(10) (a) Kirby, A. J ; Mujahid, T. G.; Camilleri, P. J. Chem. Soc., Perkin
`Trans. 2 1979, 1610. (b) Camilleri, P.; Ellul, R.; Kirby, A.; Mujahid, T. G.
`J. Chem. Soc., Perkin Trans. 2 1979, 1617. (c) Fife, T. H ; Duddy, N. W.
`J. Am. Chem. Soc. 1983, 105, 74 and reference therein.
`(11) Fersht, A. R.; Requena, Y. J. Am. Chem. Soc. 1971, 93, 3499.
`(12) Morawetz, H.; Otaki, P. S. J. Am. Chem. Soc. 1963, 85, 436.
`(13) (a) Guthrie, J. P. J. Am. Chem. Soc. 1974, 96, 3608. (b) Guthrie, J.
`P.; Pike, D. C.; Lee, Y. Can. J. Chem. 1992, 70, 1671.
`
`10.1021/ja990104d CCC: $18.00 © 1999 American Chemical Society
`Published on Web 05/01/1999
`
`SENJU EXHIBIT 2073
`LUPIN v SENJU
`IPR2015-01105
`
`PAGE 1 OF 10
`
`

`
`Equilibrium Formation of Anilides
`
`J. Am. Chem. Soc., Vol. 121, No. 19, 1999 4599
`
`us14 have shown that it is possible, under certain conditions, to
`form substantial amounts of simple amides from intermolecular
`reaction of their constituent acids and amines in water either
`with or without catalysis. In view of the attractiveness of forming
`the amide bond under “green” conditions that avoid the use of
`acyl activating agents and dry, non-hydroxylic solvents, we have
`embarked upon a program to investigate the scope and limita-
`tions of the aqueous amide bond formation so as to define the
`potential applications. The following reveals our findings for
`the formation of simple anilides in aqueous acidic media under
`acidic conditions.
`
`Experimental Section
`
`A. Materials and General Methods. Aniline, formic acid, and
`p-nitroaniline were obtained from BDH. Acetanilide, formanilide, acetic
`anhydride, and p-methoxyaniline were purchased from Aldrich and used
`as supplied. Glacial acetic acid was obtained from Fisher Scientific.
`Methanol (HPLC grade) was obtained from EM Science. Aniline and
`acetic acid were distilled prior to use. All HPLC solvents were filtered
`through a 0.45 (cid:237)m filter before use. All melting points were obtained
`using Fisher-Johns melting point apparatus and are uncorrected.
`All buffers were prepared using purified deoxygenated water from
`an Osmonic Aries water purification system. The pH was measured at
`ambient temperature using a Radiometer Vit 90 video titrator equipped
`with a GK2321 C combination electrode, standardized by Fisher
`Certified pH 2, 4, 7, and 10 buffers.
`B. Synthesis. p-Nitroformanilide and p-methoxyformanilide were
`synthesized from p-nitroaniline and p-methoxyaniline and formic acetic
`anhydride as described15 by Krishnamurthy. p-Nitroformanilide was
`recrystalized 2 times from ethyl acetate before use and had a melting
`point of 196-197 (cid:176)C (lit. 16 mp 196-198 (cid:176)C). p-Methoxyformanilide
`was purified by recrystalizing 3 times from a chloroform-hexane
`mixture. The pure product had a melting point of 79-80 (cid:176)C (lit. 17 mp
`78-80 (cid:176)C).
`C. Analytical HPLC Conditions. For HPLC analysis a Hewlett-
`Packard 1050 series HPLC system, fitted with a variable-wavelength
`UV-vis detector and autoinjector, was used. For separation, an
`(cid:237)-Bonda-Pak C18 (Waters) cartridge column was used. A gradient
`mixture of 0.005 M potassium phosphate buffer (pH 7.2) and methanol
`was used to separate aniline from formanilide. For each injection the
`initial solvent composition was 20% methanol:80% phosphate buffer,
`which after 15 min was modified to 30% methanol:70% phosphate
`buffer until 21 min, whereupon 100% methanol was used to wash the
`column for 9 min before the next injection. For separation of aniline
`and acetanilide, an isocratic mixture of 25% methanol:75% phosphate
`buffer (0.005 M, pH 7.2) was used. In both cases, the flow rate was
`1.2 mL/min and the detector was set at (cid:236) ) 231 nm.
`D. Kinetics of Amide Formation and Hydrolysis by HPLC. For
`kinetics studies at 98 ( 2 (cid:176) C, (5.0-5.3) (cid:2) 10-3 M formanilide or aniline
`solutions were made in 10-20 mL of formate buffer (pH 3.2-4.2,
`[buffer]total ) 0.1-1.0 M, (cid:237) ) 1.0 M (KCl)) and were degassed by
`passing Ar through them for 30 min. These solutions were then divided
`into 10-20 autosampler vials that were then sealed with Teflon-lined
`septa. The vials were heated in a boiler containing boiling water, and
`at certain intervals, the vials were withdrawn, cooled immediately in
`ice water, and analyzed at ambient temperature by HPLC. The pseudo-
`first-order rate constants for appearance and disappearance of aniline
`and formanilide were obtained by NLLSQ fitting of peak area vs time
`data to a standard exponential model (vide supra). The response factors
`for formanilide and aniline under the experimental conditions were
`
`(14) Keillor, J. W.; Neverov, A. A.; Brown, R. S. J. Am. Chem. Soc.
`1994, 116, 4669.
`(15) Krishnamurthy, S. Tetrahedron. Lett. 1982, 23, 3315.
`(16) Perrin, C. L.; Thoburn, J. D.; Kresge, A. J. J. Am. Chem. Soc. 1992,
`114, 8800.
`(17) DeWolfe, R.; Newcomb, C. R. J. Org. Chem. 1971, 36, 3870.
`
`obtained by injecting, and determining the peak areas of, a 1:1
`formanilide and aniline mixture. For each run two rate constants could
`be obtained, for example, in the hydrolysis direction, the pseudo-first-
`order rate constant of disappearance of the formanilide as well as the
`rate constant for the appearance of aniline.
`Kinetic data for formation and hydrolysis of acetanilide were
`obtained similarly at 98 ( 2 (cid:176)C, pH 1.95 ( 0.03 and pH 3.72 ( 0.03
`with (5.2-5.4) (cid:2) 10-3 M solutions of acetanilide or aniline, (cid:237) ) 1.0
`(KCl). At lower pH, HCl comprised the buffer and 1.0 M acetic acid
`was added to the solutions. At higher pH, acetate buffer was used with
`the total [buffer] being 1.0 M.
`E. Kinetics by UV-Vis Spectrophotometry. The rates of formation
`and hydrolysis of formanilide, p-nitroformanilide, and p-methoxyfor-
`manilide were observed at 79 ( 1 (cid:176)C using a Cary-219 UV -vis
`spectrophotometer interfaced with an IBM 486 PC equipped with Olis
`software (Online Instrument Systems, Jefferson GA, 1992). Kinetic data
`were obtained by observing the rate of change in absorbance (increase
`for formation and decrease for hydrolysis) of formanilide at 246 nm
`and p-methoxyformanilide at 260 nm. For p-nitroformanilide, rates of
`formation and hydrolysis were obtained by following the rates of
`disappearance and appearance of p-nitroaniline, respectively, at 429
`nm. Runs were initiated by injecting 5 (cid:237)L of a stock solution in DME
`((5.0-6.9) (cid:2) 10-2 M) of either (p-H, p-NO2, or p-OCH3)-aniline or
`(p-H or p-NO2 or p-OCH3)-formanilide into 3 mL of formate buffer
`(pH 2.80-4.20, [buffer]total ) 0.001-1.0 M, (cid:237) ) 1.0 (KCl)), which
`had been thermally equilibrated at 79 ( 1 (cid:176)C in the instrument cell
`holder for 30 min. The final pH of each of the runs was measured and
`shown to agree with the initial pH. The pseudo-first-order rate constants
`were obtained by NLLSQ fitting the absorbance vs time data to a
`standard exponential model (At ) A¥ + (A0 - A¥ ) exp(-kt)).
`The acid-catalyzed hydrolysis of formanilide was followed at 79 (
`1 (cid:176) C using 9.86 (cid:2) 10-3 M HCl, (cid:237) ) 1.0 (KCl), with runs being initiated
`as above. All the runs were followed for at least 5 half-lives.
`F. D2O Studies. The rate of formation and hydrolysis of formanilide
`at 79 ( 1 (cid:176)C was determined in formate buffer ([buffer] total ) 0.25-
`1.0 M) at pD 3.60 ( 0.03 where pD ) pHmeasured + 0.40.18 The rate of
`hydrolysis of formanilide was also obtained in 1.10 (cid:2) 10-2 M DCl, (cid:237)
`) 1.0 (KCl). The kinetic data were obtained and analyzed as above.
`G. Studies in the Presence of Phosphate. The rate of formation
`and hydrolysis of formanilide was determined similarly at 79 ( 1 (cid:176)C
`in the presence of phosphate (0.10-0.50 M) in 1.0 M formate buffer
`at pH 3.20 ( 0.03 and pH 3.60 ( 0.03, with the ionic strength again
`being maintained at 1.0 (KCl).
`H. Studies in Aqueous Ethanol. The rate of formation and
`hydrolysis of formanilide was obtained in 1.0 M formate buffer in 20%
`(v/v) ethanolic water, pH 3.59, (cid:237) ) 1.0 (KCl). The kinetic data were
`obtained as above at 60 ( 0.3 (cid:176)C. Kinetic data were also obtained in
`80% (v/v) ethanolic water containing 1.0 M formate at pHs 3.60 and
`4.92 at the same temperature. The pH was adjusted by adding a suitable
`amount of concentrated NaOH or HCl and measured before and after
`the reaction. The ionic strength in 80% ethanol-20%water was not
`corrected. The rate of formation and hydrolysis of formanilide was also
`obtained at 60 (cid:176)C in 1.0 M aqueous formate buffer pH 3.60, (cid:237) ) 1.0
`(KCl).
`I. Determination of pKa. The pKa’s of aniline and formic acid were
`determined by titration at 24 ( 1 (cid:176)C. For each determination, 0.048 -
`0.051 mmol of aniline or formic acid was used in a 5 mL solution. In
`the case of aniline enough HCl was added to convert all of it to
`anilinium ion. The pH was measured using a Radiometer Vit 90 video
`titrator equipped with a GK2321C combination electrode and interfaced
`with an IBM PC. The pH was recorded as a function of added 0.0105
`M NaOH, which was delivered by a Radiometer ABU 91 autoburet.
`The ionic strengths of all of the solutions were maintained at 1.0 using
`KCl. Data were analyzed by a computer version of Simms method.19
`The pKa values reported are an average of three determinations.
`Reported in Table 10 (as calculated in the Appendix) are pKa values
`corrected for the temperature and medium effects.
`
`(18) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188.
`(19) Huguet, J. Ph.D. Thesis, University of Alberta, 1980
`
`PAGE 2 OF 10
`
`

`
`4600 J. Am. Chem. Soc., Vol. 121, No. 19, 1999
`
`Aman and Brown
`
`constant, K¢, is given in eq 3.
`
`+ kr
`) kf
`kobs
`K¢ ) kf/kr
`
`(2)
`
`(3)
`
`In practical terms, K¢
`is the ratio of [anilide]/[aniline]total at
`equilibrium with a given excess of RCOOH. Given in Tables
`1S-12S (Supporting Information) are the original kobs data for
`appearance or disappearance of various the anilides as well as
`the conditional equilibrium constants, K¢, determined at equi-
`librium by HPLC methods or from the residual absorbance in
`the absorbance vs time UV kinetic plots. Given in Tables 1-8
`are the pseudo-first-order rate constants for formation (kf) and
`hydrolysis (kr) of the anilides as well as the corrected equilibrium
`constant, K¢ eq, which is based on the concentrations of nonion-
`ized aniline and formic acid with the concentration of water set
`as unity as in eq 4.2b The acid and amine concentrations can be
`
`K¢
`
`eq
`
`)
`
`[formanilide]
`[aniline][formic acid]
`
`(4)
`
`Figure 1. Typical kinetic traces for formanilide equilibration at pH
`3.60 at different [formate]total, 0.10-1.00 M, (cid:237) ) 1.0 (KCl), and T )
`79 ( 1 (cid:176)C. The lines are obtained from NLLSQ fitting of data to At )
`A¥ + (A0 - A¥ ) exp(-kt). formation at 0.1 M (9), hydrolysis at 0.1 M
`(0), formation at 0.5 M (2), hydrolysis at 0.5 M (1), formation at 1.0
`M ([), and hydrolysis at 1.0 M (b).
`
`Scheme 1
`
`determined from the pKa values for the various RCOOH and
`Ar-NH3
`+, corrected for the conditions of temperature and
`media as described in the Appendix, the corrected values being
`listed in Table 10. We deal with the specific conditions of
`temperature and structure below, followed by the effects of
`Results
`[formate] and additives.
`The approach to equilibrium for the anilides given in Scheme
`A. Effect of Temperature and Structure on Anilide
`1 was followed both from the direction of formation and
`Equilibrium Constants. Given in Tables 1-3 are the experi-
`hydrolysis under a variety of conditions such as temperature,
`mental kf, kr, and K¢ eq values for equilibration of formanilide
`pH, [RCOOH]total, and the presence of the additives ethanol and
`and acetanilide at various pHs and [RCOOH]total at the indicated
`phosphate. Shown in Figure 1 are typical absorbance vs time
`temperatures. The data show that the K¢ eq for formanilide at 98
`traces for the equilibration of formanilide with aniline and
`(cid:176)C (12.6 M -1) is larger than that for acetanilide (3.4 M-1) by
`formate at pH 3.60 and 79 (cid:176)C in the presence of three different
`a factor of about 3.7. As expected, equilibrium is reached faster
`[formate]total. The pseudo-first-order rate constant for the ap-
`at higher temperature, but reducing the temperature from 98 to
`proach to equilibrium (kobs) can be obtained by NLLSQ fitting
`79 (cid:176) C leads to an increase in the K¢ eq for formanilide of roughly
`of the absorbance vs time data to a standard exponential model,
`1.6-fold, (12.6 vs 20.1 M-1). Given in Table 3 are data
`At ) A¥ + (A0 - A¥ ) exp(-kt). The kobs can be expressed in
`determined in D2O at 79 (cid:176)C that indicate that, at pH (pD) 3.60,
`terms of the forward and reverse rate constants, kf and kr
`the overall solvent deuterium kinetic isotope effect (DKIE) on
`(Scheme 1) as in eq 2, while the conditional equilibrium
`Table 1. Pseudo-First-Order Rate Constantsa for Formation (kf) and Hydrolysis (kr) of Formanilide at 98 ( 2 (cid:176)C and the Equilibrium
`Constantsb (K¢ eq) Obtained by HPLC at Various pHs and Buffer Concentrations in Aqueous Formate Buffer
`K¢ eq (M-1)
`kr (s-1)
`kf (s-1)
`pH
`c (M)
`[formate]total
`(0.23 ( 0.01) (cid:2) 10-5
`(1.22 ( 0.03) (cid:2) 10-5
`12.3 ( 0.2
`4.17 ( 0.03
`0.10
`(4.15 ( 0.02) (cid:2) 10-5
`(1.13 ( 0.04) (cid:2) 10-5
`3.60 ( 0.03
`13.1 ( 0.6
`0.10
`(1.29 ( 0.06) (cid:2) 10-4
`(2.56 ( 0.11) (cid:2) 10-5
`3.18 ( 0.03
`12.9 ( 0.2
`0.10
`(9.72 ( 0.30) (cid:2) 10-5
`(1.20 ( 0.01) (cid:2) 10-4
`3.57 ( 0.03
`11.9 ( 0.4
`0.50
`(1.27 ( 0.04) (cid:2) 10-4
`(3.36 ( 0.08) (cid:2) 10-4
`3.57 ( 0.03
`12.7 ( 0.2
`1.00
`a Calculated from the pseudo-first-order rate constants (kobs) for establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors are
`deviation from the mean for duplicate values and standard deviations for triplicate values. b Calculated using the nonionized concentrations at
`equilibrium (eq 4), taking concentration of water as unity and accounting for changes in the pKa’s due to temperature and ionic strength variations.
`c (cid:237) ) 1.0 (KCl).
`Table 2. Pseudo-First-Order Rate Constantsa for Formation (kf) and Hydrolysis (kr) of Acetanilide at 98 ( 2 (cid:176)C and the Equilibrium
`Constantsb (K¢ eq) at Various pH Values in Aqueous HCl or Acetate Buffer
`K¢ eq (M-1)
`kf (s-1)
`kr (s-1)
`pH
`buffer
`(0.19 ( 0.02) (cid:2) 10-5
`(3.47 ( 0.04) (cid:2) 10-5
`3.6 ( 0.3
`1.95 ( 0.05
`HClc e
`(1.07 ( 0.02) (cid:2) 10-6
`(0.81 ( 0.02) (cid:2) 10-6
`3.75 ( 0.05
`3.2 ( 0.3
`acetated e
`a Calculated from the pseudo-first-order rate constants (kobs) for establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors are
`deviation from the mean for duplicate values and standard deviations for triplicate values. b Calculated using the nonionized concentrations at
`equilibrium, taking concentration of water as unity and accounting for changes in the pKas due to temperature and ionic strength variations. c 1.0
`M acetate added. d [buffer]total ) 1.0 M. e (cid:237) ) 1.0 (KCl).
`
`PAGE 3 OF 10
`
`

`
`K¢ eq (M-1)
`20.6
`22.7
`20.7
`19.1
`18.2
`18.6
`
`J. Am. Chem. Soc., Vol. 121, No. 19, 1999 4601
`Equilibrium Formation of Anilides
`Table 3. Pseudo-First-Order Rate Constantsa for Formation (kf) and Hydrolysis (kr) of Formanilide at 79 ( 1 (cid:176)C and the Equilibrium
`Constantsb (K¢ eq) at Various pHs or pDs and at Various Buffer Concentrations in Aqueous Formate Buffer
`Kr (s-1)
`kf (s-1)
`pH or pD
`d (M)
`[formate]total
`(0.77 ( 0.01) (cid:2) 10-5
`(7.02 ( 0.05) (cid:2) 10-5
`2.80 ( 0.03
`0.10
`(7.60 ( 0.10) (cid:2) 10-5
`(4.29 ( 0.05) (cid:2) 10-5
`2.79 ( 0.03
`0.50
`(7.69 ( 0.02) (cid:2) 10-5
`(8.22 ( 0.02) (cid:2) 10-5
`2.81 ( 0.03
`0.90
`(3.32 ( 0.05) (cid:2) 10-5
`(0.66 ( 0.02) (cid:2) 10-5
`3.18 ( 0.03
`0.10
`(3.66 ( 0.05) (cid:2) 10-5
`(3.60 ( 0.05) (cid:2) 10-5
`3.20 ( 0.03
`0.50
`(4.09 ( 0.03) (cid:2) 10-5
`(8.20 ( 0.02) (cid:2) 10-5
`3.20 ( 0.03
`1.00
`(1.36 ( 0.02) (cid:2) 10-5
`3.56 ( 0.03
`0.001
`(1.42 ( 0.02) (cid:2) 10-5
`(0.42 ( 0.01) (cid:2) 10-5
`3.60 ( 0.03
`0.10
`(1.76 ( 0.03) (cid:2) 10-5
`(2.64 ( 0.02) (cid:2) 10-5
`3.61 ( 0.03
`0.50
`(2.15 ( 0.03) (cid:2) 10-5
`(5.97 ( 0.04) (cid:2) 10-5
`3.59 ( 0.03
`1.00
`(5.93 ( 0.02) (cid:2) 10-6
`(1.83 ( 0.01) (cid:2) 10-6
`4.01 ( 0.03
`0.10
`(7.90 ( 0.01) (cid:2) 10-6
`(1.25 ( 0.01) (cid:2) 10-5
`4.02 ( 0.03
`0.50
`(1.06 ( 0.01) (cid:2) 10-5
`(2.94 ( 0.01) (cid:2) 10-5
`4.03 ( 0.03
`1.00
`(1.34 ( 0.03) (cid:2) 10-5
`3.60 ( 0.03c
`0.001
`(1.93 ( 0.09) (cid:2) 10-5
`(1.77 ( 0.10) (cid:2) 10-5
`3.58 ( 0.03c
`22.3
`0.50
`(2.01 ( 0.12) (cid:2) 10-5
`(2.76 ( 0.30) (cid:2) 10-5
`3.61 ( 0.03c
`21.9
`0.75
`(2.18 ( 0.03) (cid:2) 10-5
`(3.96 ( 0.06) (cid:2) 10-5
`3.60 ( 0.03c
`21.6
`1.00
`a Calculated from the pseudo-first-order rate constants (kobs) for establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors are
`deviation from the mean for duplicate values and standard deviations for triplicate values. b Calculated using the nonionized concentrations at
`equilibrium and taking concentration of water as unity. Changes in the pKa’s due to temperature, ionic strength, and solvent isotope effects have
`been considered. c pD. d (cid:237) ) 1.0 (KCl).
`Table 4. Pseudo-First-Order Rate Constantsa for Formation (kf) and Hydrolysis (kr) ofp-Nitroformanilide at 79 ( 1 (cid:176)C and the Equilibrium
`Constantsb (K¢ eq) at Various pHs and Buffer Concentrations in Aqueous Formate Buffer
`K¢ eq (M-1)
`kf (s-1)
`kr (s-1)
`pH
`c (M)
`[formate]total
`(5.34 ( 0.04) (cid:2) 10-5
`(2.33 ( 0.05) (cid:2) 10-4
`2.80 ( 0.03
`0.26
`1.00
`(9.86 ( 0.02) (cid:2) 10-5
`(2.16 ( 0.20) (cid:2) 10-6
`3.18 ( 0.03
`0.10
`0.29
`(1.12 ( 0.03) (cid:2) 10-4
`(1.17 ( 0.05) (cid:2) 10-5
`3.20 ( 0.03
`0.50
`0.27
`(1.28 ( 0.03) (cid:2) 10-4
`(2.40 ( 0.06) (cid:2) 10-5
`3.20 ( 0.03
`1.00
`0.26
`(7.22 ( 0.07) (cid:2) 10-5
`(9.33 ( 0.60) (cid:2) 10-6
`3.60 ( 0.03
`1.00
`0.25
`a Calculated from the pseudo-first-order rate constants (kobs) for establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors are
`deviation from the mean for duplicate values and standard deviations for triplicate values. b Calculated using the nonionized of concentrations at
`equilibrium and taking concentration of water as unity. Changes in the pKa’s due to temperature have been considered. For formic acid, the change
`in pKa due to ionic strength variation has also been considered. However, the effect of ionic strength on the pKa of p-nitroanilinium ion was not
`considered since at these pHs, a change of (0.35 in pKa unit will have less than 2% effect on the K¢ eq. c (cid:237) ) 1.0 (KCl).
`
`20.4
`20.9
`19.5
`20.9
`21.7
`19.0
`
`K¢ eq (M-1)
`41.0
`38.9
`
`Table 5. Pseudo-First-Order Rate Constantsa for Formation (kf)
`and Hydrolysis (kr) ofp-Methoxyformanilide at 79 ( 1 (cid:176)C and the
`Equilibrium Constantsb (K¢ eq) at pHs 2.80 and 3.20 in Aqueous
`Formate Buffer
`kf (s-1)
`kr (s-1)
`pHc
`(4.01 ( 0.04) (cid:2) 10-5
`2.82 ( 0.03 (4.08 ( 0.05) (cid:2) 10-5
`3.18 ( 0.03 (2.13 ( 0.03) (cid:2) 10-5
`(4.11 ( 0.06) (cid:2) 10-5
`a Calculated from the pseudo-first-order rate constants (kobs) for
`establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors
`are deviation from the mean for duplicate values and standard deviations
`for triplicate values. b Calculated using the nonionized of concentrations
`at equilibrium and taking concentration of water as unity and accounting
`for changes in the pKa’s due to temperature and ionic strength.
`c [buffer]total ) 1.0, (cid:237) ) 1.0 (KCl).
`the K¢ eq for formanilide is 1.1 ( 0.1 but that the DKIE on K¢ )
`kf/kr is 1.5-1.6 ([formate]total ) 0.5-1.0 M).
`Given in Tables 4 and 5 are the kf, kr, and K¢ eq values for the
`equilibration of p-nitroformanilide and p-methoxyformanilide
`at 79 (cid:176)C at various pHs. The respective K¢ eq values of 0.26 and
`40 M-1, coupled with the value of 20.1 M-1 for formanilide at
`79 (cid:176) C, indicate a strong dependence on the basicity of the aniline
`substituent.
`B. Effect of [formate] on kf and kr. Shown in Figure 2 are
`typical plots of the pseudo-first-order rate constants for forma-
`tion (kf) and hydrolysis (kr) of formanilide as a function of
`[formate]total at 98 (cid:176)C, pH ) 3.60, and (cid:237) ) 1.0 (KCl). Similar
`plots (not shown) can be made for the processes at 79 (cid:176)C. The
`data indicate that the hydrolysis follows a linear dependency
`on the [formate], while the formation follows an exponential
`behavior. The kinetic/concentration data for the two processes
`
`Figure 2. Pseudo-first-order rate constants of formation (kf, 2) and
`hydrolysis (kr, 9) of formanilide vs [formate]total at 98 ( 2 (cid:176)C (pH )
`3.60 and (cid:237) ) 1.0 (KCl)). The lines were obtained from the fits to eqs
`5 and 6.
`
`can be fit to eqs 5 and 6,
`+ k1r[formate]total
`) k0r
`kr
`) [formate]total(k1f
`+ k2f[formate]total)
`
`kf
`
`(5)
`
`(6)
`
`where k0r, the intercept for eq 5, is the spontaneous pseudo-
`first-order rate constant for hydrolysis at pH 3.60, k1r is the
`second-order rate constant for formate-catalyzed hydrolysis, and
`k1f and k2f are the second- and third-order rate constants for
`formate-dependent hydrolysis and formation of formanilide.
`Fitting of the appropriate hydrolysis and formation data at 98
`and 79 (cid:176) C (from Tables 1 and 3) to eqs 5 and 6 gives the values
`
`PAGE 4 OF 10
`
`

`
`4602 J. Am. Chem. Soc., Vol. 121, No. 19, 1999
`
`Aman and Brown
`
`pH or pD
`2.80
`3.20
`3.60
`4.00
`3.60c d
`2.00d
`1.96c
`3.60
`
`Table 6. Pseudo-First-Order Rate Constants for the Acid Catalyzed Hydrolysis, k0r (s-1),a of Formanilide at a Given pH or pD, Second-Order
`Rate Constants, k1r (M-1 s-1),a of Formate (buffer) Catalysis on Hydrolysis, and Second- and Third-Order Rate Constants, k1f (M-1 s-1)a and k2f
`(M-2 s-1),a of Formate (buffer) Catalysis on Formation of Formanilide from Aniline and Formic Acid at Various Temperatures and pHs or pDs
`k0r(cid:2) 105
`k1r (cid:2) 106
`K1f (cid:2) 105
`k2f (cid:2) 105
`temp ((cid:176)C)
`(s-1)
`(M-1 s-1)
`(M-1 s-1)
`(M-2 s-1)
`7.84 ( 0.09
`1.45 ( 0.11
`7.02 ( 0.21
`8.50 ( 3.50b
`79 ( 1
`3.24 ( 0.01
`8.45 ( 0.01
`6.24 ( 0.05
`1.95 ( 0.06
`79 ( 1
`1.35 ( 0.01
`8.01 ( 0.14
`4.45 ( 0.14
`1.47 ( 0.15
`79 ( 1
`0.54 ( 0.01
`5.19 ( 0.13
`2.02 ( 0.08
`0.93 ( 0.08
`79 ( 1
`79 ( 1
`1.38 ( 0.08
`8.31 ( 1.10
`3.04 ( 0.10
`0.91 ( 0.10
`79 ( 1
`43.8 ( 0.01
`79 ( 1
`53.2 ( 0.01
`19.9 ( 0.20
`13.7 ( 0.10
`95.0 ( 18.0
`3.72 ( 0.56
`98 ( 2
`a The errors calculated from the standard deviation of the fit of kr vs [formate]total and kf vs [formate]total to eqs 5 and 6, respectively, at the given
`pH or pD. b At this pH the acid-catalyzed hydrolysis, k0r, is much higher than the buffer catalyzed hydrolysis, k1r (less than 11% at highest buffer
`concentration). c pD. d HCl or DCl was used as buffer; no formate added, (cid:237) ) 1.0 (KCl).
`Table 7. Pseudo-First-Order Rate Constants of Formation (kf) and Hydrolysis (kr) of Formanilide and the Equilibrium Constants (K¢ eq) in the
`Presence of Phosphate at 79 ( 1 (cid:176)C in Aqueous Formate Buffer and the Second-Order Rate Constant for Phosphate Catalysis on Hydrolysis
`phos)b
`phos)b and Formation (kf
`(kr
`phos (cid:2) 105 (M-1 s-1)
`phos (cid:2) 105 (M-1 s-1)
`kr (cid:2) 105 (s-1)
`kf (cid:2) 105 (s-1)
`K¢ eq (M-1)
`pHa
`[phosphate]total (M)
`(4.66 ( 0.02)
`(9.50 ( 0.03)
`6.14 ( 0.05
`13.4 ( 0.10
`18.5
`3.21
`0.10
`(5.90 ( 0.10)
`(12.2 ( 0.05)
`18.6
`3.21
`0.30
`(2.47 ( 0.03)
`(6.71 ( 0.02)
`19.1
`3.58
`0.10
`(3.52 ( 0.05)
`(9.44 ( 0.01)
`18.8
`3.60
`0.50
`a [buffer]total ) 1.00 M, (cid:237) ) 1.0 (KCl). b The errors are calculated from the standard deviation of the fit of kr vs [phosphate]total and kf vs
`[phosphate]total to a linear equation, kr ) kr
`0 + kr
`phos[phosphate]total or kf ) kf
`0 + kf
`0 are the pseudo-first-order rate
`phos[phosphate]total, where kr
`0 and kf
`constants of hydrolysis and formation without added phosphate under the experimental conditions at the given pH. The data at zero phosphate
`concentration were obtained from Table 4.
`Table 8. Pseudo-First-Order Rate Constantsa of Formation (kf) and Hydrolysis (kr) of Formanilide and the Equilibrium Constantsb (K¢ eq) in
`Aqueous Ethanolic, Formate Buffer at 60.0 ( 0.3 (cid:176)C
`% (v/v)
`K¢ eq (M-1)d
`kf (s-1)
`kr (s-1)
`pHc
`ethanol
`(3.30 ( 0.02) (cid:2) 10-5
`(5.32 ( 0.55) (cid:2) 10-6
`3.59 ( 0.03
`23.9
`20
`(2.82 ( 0.03) (cid:2) 10-6
`(1.85 ( 0.02) (cid:2) 10-5
`4.92 ( 0.05
`80
`14.4e
`(7.14 ( 0.02) (cid:2) 10-5
`(7.01 ( 0.02) (cid:2) 10-6
`3.60 ( 0.05
`9.25e
`80
`(1.65 ( 0.02) (cid:2) 10-5
`(5.86 ( 0.03) (cid:2) 10-6
`3.60 ( 0.05
`32.7
`0
`a Calculated from the pseudo-first-order rate constants (kobs) for establishment of equilibrium, using kobs ) kr + kf and K¢ ) kf/kr. Errors are
`deviations from the mean for duplicate values and standard deviations for triplicate values. b Calculated using the nonionized of concentrations at
`equilibrium and taking concentration of water as unity, accounting for the changes in the pKa’s due to the percent ethanol present in the solvent and
`variation of temperature. 20% v/v ethanol water corresponds to 16.4 wt %, and 80% v/v corresponds to 75.8 wt %. c [buffer]total ) 1.0 M. d Given
`the possibility that ethyl formate is produced during the reaction, that species could be involved in the formation of formanilide, and that the water
`concentration can no longer be taken as unity, the K¢ eq should be written as K¢ eq) ([amide][H2O])/(([acid] + [ethyl formate])[amine]). e (cid:237) uncorrected.
`
`kr
`
`kf
`
`2.70 ( 0.09
`
`6.90 ( 0.09
`
`for these rate constants compiled in Table 6. Two things are
`apparent from the data in Table 6. First, the k0r constants increase
`linearly with increasing [H3O+], indicating that the hydrolysis
`in the absence of buffer is specific acid catalyzed. Second, from
`D+) ratio at pH
`H+/k0r
`the D2O data given in the table, the (k0r
`(pD) 3.6 is 1.0 ( 0.1, while that at pH 2.0 is 0.9. The solvent
`DKIE on the buffer catalysis for hydrolysis at pH (pD) 3.6,
`D2O), is 0.96 ( 0.13, while the DKIE for formation
`H2O/k1r
`(k1r
`D2O) ) 1.61 (
`D2O) ) 1.46 ( 0.10 and (k2f
`H2O/k1f
`H2O/k2f
`are (k1f
`0.31.
`C. Effect of Additives on the kf, kr, and K¢ eq Constants.
`Given in Table 7 are the kf, kr, and K¢ eq constants for the
`equilibration of formanilide at 79 (cid:176)C at pHs 3.20 and 3.60 in a
`1.0 M formate buffer in the presence of K2PO4. At these pH
`values, the phosphate exists as the monoanionic form and the
`ionic strength was held at 1.0 with added KCl. Although
`equilibrium is attained faster in the presence of phosphate, the
`K¢ and K¢ eq values are unchanged relative to those obtained in
`the absence of phosphate. Plots (not shown) of the kf and kr
`values against [phosphate] give straight lines, the slopes of which
`phos) and
`give the phosphate catalysis on the formation (kf
`phos) processes: these values are also listed in Table
`hydrolysis (kr
`7.
`
`Given in Table 8 are the pseudo-first-order rate constants for
`formation and hydrolysis of formanilide in aqueous ethanolic
`solutions of [formate]total ) 1.0 M, T ) 60 (cid:176) C, and ionic strength
`not held constant. In keeping with the trends of temperature on
`K¢ eq noted in part A above, in the absence of alcohol, the
`corrected equilibrium constant increases at lower temperature
`although the rate of attainment of equilibrium decreases. Added
`ethanol has the effect of decreasing both kr and kf, but by
`different amounts, the net effect being to reduce K¢ eq as the
`ethanol content increases.
`
`Discussion
`A summary of the condit