Metallomicelles Made of Ni(II) Complexes of Lipophilic 2

2-Pyridineketoximes as Powerful Catalysts of the Cleavage of Carboxylic Acid ... Umberto Tonellato*,‡. Department of Organic ... and Centro CNR Mecc...
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Langmuir 1999, 15, 405-412

405

Metallomicelles Made of Ni(II) Complexes of Lipophilic 2-Pyridineketoximes as Powerful Catalysts of the Cleavage of Carboxylic Acid Esters Frantisek Hampl,† Frantisek Liska,† Fabrizio Mancin,‡ Paolo Tecilla,*,‡ and Umberto Tonellato*,‡ Department of Organic Chemistry, Prague Institute of Chemical Technology, Technicka´ 5, 16628 Praha 6, Czech Republic, and University of Padova, Department of Organic Chemistry and Centro CNR Meccanismi di Reazione Organiche, Via Marzolo,1 35131 Padova, Italy Received July 10, 1998. In Final Form: October 13, 1998 A series of 2-pyridineketoximes with paraffinic chains of different lengths (CH3, 1a; C8H17, 1b; C13H27, 1c) has been synthesized, and their complexes with Cu(II), Co(II), Zn(II), and Ni(II) have been investigated in the cleavage of p-nitrophenyl esters of carboxylic and phosphoric acids in water (1a) or in comicelles with CTABr (1b,c). While the Co(II) and Cu(II) complexes are ineffective in promoting the cleavage of acetate (PNPA) and hexanoate (PNPH) esters, the Zn(II) and especially the Ni(II) complexes strongly accelerate the cleavage of such substrates. With the latter metal ion, the effective species is a complex with a 2:1 ligand/metal ion stoichiometry and a pKa of the oximic hydroxyl of approximately 5 and 3.7 in the absence and in the presence of CTABr, respectively. Strong evidence has been obtained concerning the mode of action which involves nucleophilic attack of the oximate function on the carbonyl carbon of the ester to give, as transient intermediate, the acylated oxime; its Ni(II)-mediated hydrolysis by water has also been investigated to better define the whole catalytic cycle. The system displays its highest efficiency at low pH values and in comicelles of CTABr: in the presence of 1b/Ni(II)/CTABr at pH 4 the observed rate enhancements are of over 5 orders of magnitude in the cleavage of PNPH and the system is truly catalytic with a sizable turnover rate. In comparison with the high reactivity toward carboxylic esters, the metal ion complexes of the ketoximes investigated are surprisingly ineffective in promoting the cleavage of p-nitrophenyl esters of diphenylphosphoric acid (PNPDPP).

Introduction In recent years, transition metal ions complexes with lipophilic ligands in micellar or vesicular aggregates have attracted considerable attention as catalysts of the cleavage of esters and amides and as biomimetic models of hydrolytic metalloenzymes.1 The structure of the most effective ligands so far investigated features an hydroxy function located in the proximity of the chelating subsite so that it may be activated through coordination with the metal ion. In the search for new and effective catalysts we turned our attention to oxime-functionalized ligands, stimulated also by the well-recognized effectiveness of pyridinium carbaldoximate ions as acetylcholinesterase reactivators.2 These reactivators, 2-[(hydroxymino)methyl]-1-methylpyridinium iodide, currently named PAM, being among the most efficient ones currently used † ‡

Prague Institute of Chemical Technology. University of Padova.

(1) (a) Gellman, S. H.; Petter, R.; Breslow, R. J. Am. Chem. Soc. 1986, 108, 2388. (b) Menger, F. M.; Gan, L. H.; Johnson, E.; Durst, D. H. J. Am. Chem. Soc. 1987, 109, 2800. (c) Fornasier, R.; Scrimin, P.; Tecilla, P.; Tonellato, U. J. Am. Chem. Soc. 1989, 111, 224. (d) Weijnen, J. G. J.; Koudijs, A.; Engbersen, J. F. J J. Org. Chem. 1992, 57, 7258. (e) Ogino, K.; Kashihara, N.; Ueda, T.; Isaka, T.; Yoshida, T.; Tagaki, W. Bull. Chem. Soc. Jpn. 1992, 65, 373. (f) Scrimin, P.; Tecilla, P.; Tonellato, U. J. Org. Chem. 1991, 56, 161. (g) Scrimin, P.; Tecilla, P.; Tonellato, U. J. Am. Chem. Soc. 1992, 114, 5086. (h) Scrimin, P.; Tecilla, P.; Tonellato, U. J. Org. Chem. 1994, 59, 18. (i) Bunton, C. A.; Scrimin, P.; Tecilla, P. J. Chem. Soc., Perkin Trans. 2 1996, 419. (j) Kriste, A. G.; Vizitiu, D.; Thatcher, R. J. Chem. Commun. 1996, 913. (k) Scrimin, P.; Tecilla, P.; Moss, R. A.; Bracken, K. J. Am. Chem. Soc. 1998, 120, 1179. (2) (a) Bedford, C. D.; Harris, R. W.; Howd, R. A.; Miller, A.; Nolen, H. W., III; Kenley, R. A. J. Med. Chem. 1984, 27, 1431. (b) Kenley, R. A.; Bedford, C. D.; Dailey, O. D.; Howd, R. A.; Miller, A. J. Med. Chem. 1984, 27, 1201. (c) Bedford, C. D.; Miura, M.; Bottaro, J. C.; Howd, R. A.; Nolen, H. W., III. J. Med. Chem. 1986, 29, 1689.

for clinical applications, act as powerful hydrolytic agents toward the phosphorylated serine of the enzyme cholinesterase poisoned by organophosphorus (nerve gas) inhibitors. The pyridinium oximes are claimed to react so effectively with the serine esters of organophosphorous acids in the enzyme (as well as with ester of carboxylic acids in vitro) because of their relatively low pKa (in the physiological pH range) that ensures, in neutral aqueous solutions, a substantial dissociation to oximate anions which are powerful (R-) nucleophiles.3 In the case of PAM and analogous substances, the low pKa is the result of the presence of the positively charged pyridinium nitrogen. Literature data indicate that an even larger effect can be achieved by chelating a transition metal ion to a 2-pyridine aldoxime or ketoxime.4 On these premises, we synthesized the 2-[(1-hydroximino)alkyl]pyridine derivatives of general structure 1 and investigated them as catalysts of the hydrolytic cleavage of esters in aqueous solutions, in the presence of micelles of cetyltrimethylammonium bromide (CTABr) in the case of the lipophilic oximes 1b,c. Micelles are known (3) Terrier, F.; MacCormack, P.; Kizilian, E.; Halle`, J.-C.; Demerseman, P.; Guir, F.; Lion, C. J. Chem. Soc., Perkin Trans. 2 1991, 153 and references cited therein. (4) (a) Orama, M.; Saarinen, H.; Korvenranta, J. Acta Chem. Scand. 1989, 43, 407. (b) Suh, J.; Cheong, M.; Suh, M. P. J. Am. Chem. Soc. 1982, 104, 1645. (c) Breslow, R.; Chipman, D. J. Am. Chem. Soc. 1965, 87, 4195. (d) Suh, J.; Suh, M. P.; Lee, J. D. Inorg. Chem. 1985, 24, 3088. (e) Suh, J.; Cheong, M.; Han, H. Bioorg. Chem. 1984, 12, 188. (5) (a) Fendler, J. Membrane Mimetic Chemistry; Wiley: New York, 1982. (b) Fendler, J. H.; Fendler, J. E. Catalysis in Micellar and Macromolecular Systems; Academic Press: New York, 1975. (c) Bunton, C. A.; Savelli, G. Adv. Phys. Org. Chem. 1986, 22, 213. (d) Fornasier, R.; Tonellato, U. J. Chem. Soc., Faraday Trans. 1980, 76, 1301. (e) Bunton, C. A.; Nome, F.; Quina, F. H.; Romsted, L. Acc. Chem. Res. 1991, 24, 357. (f) Menger, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1086.

10.1021/la980861+ CCC: $18.00 © 1999 American Chemical Society Published on Web 12/31/1998

406 Langmuir, Vol. 15, No. 2, 1999 Chart 1. Ligands and Substrates Used in This Work

Hampl et al. Table 1. Relative Rates (kψ/k0) for the Cleavage of PNPH and PNPDPP in the Presence of 1c/CTABr and Different Metal Ions {Me(II)}a kψ/k0 entry 1 2 3 4 5 6

additive CTABr 1c/CTABr 1c/CTABr 1c/CTABr 1c/CTABr 1c/CTABr

Me(II) none none Cu(II) Zn(II) Co(II) Ni(II)

PNPH 1b 18 12 210 1.7 486

PNPDPP 1c 1.4 3.5 1.3 1.3 1.3

a [1c] ) 4.12 × 10-4 M; [CTABr] ) 7.2 × 10-3 M; [Me(II)] ) 4.12 × 10-4 M; 0.05 M MES buffer, pH 6.3; T ) 25 °C. b k0 ) 3.7 × 10-6 s-1. c k0 ) 5.9 × 10-5 s-1.

to amplify the kinetic benefits by bringing together in a small volume the reactant species, and this is particularly relevant when they are markedly lipophilic and, hence, sparingly soluble in bulk solution.5 In the present case (see above), further advantage may be expected from the effect of cationic micelles which are known to lower the pKa of acidic functions.5c,d A preliminary study6 of the reactivity of compounds 1a-c in micellar aggregates in water in the presence and absence of transition metal ions pointed to a rather impressive efficacy of the micellar complexes with Ni(II) in the hydrolytic cleavage of activated (p-nitrophenyl) esters of carboxylic acids (acetate, PNPA, 3a; hexanoate, PNPH, 3b) in mildly acidic solutions and stimulated a further investigation aimed at defining the mode of action and the catalytic properties of these metalloaggregates. This is a full account of that study: (i) the amphiphilic ligands 1b,c were investigated in micellar cationic matrixes and water soluble 1a as a monomeric species; (ii) the kinetic measurements were made in the presence and absence of transition metal ions, notably Ni(II); (iii) the substrates of choice were the esters of carboxylic acids 3 (PNPA and PNPH) and of phosphoric acid (PNPDPP, 4). Compounds 2 were also prepared and investigated for comparison purposes. Moreover the study was extended to define the hydrolytic behavior of the acetylated derivatives 2b,c (Chart 1). Results Ketoximes 1 were obtained by reaction of hydroxylamine with the corresponding ketones which, in turn, were prepared from 2-pyridinecarbonitrile and the proper Grignard reagents or from ethyl picolinate via the acetoacetic synthesis. The O-methylated 2a was obtained by reaction of 2-pyridineketoxime with methoxyamine and the O-acetylated derivatives 2b,c from the corresponding oximes and acetic anhydride. Only 1a and 2a,b are moderately soluble in water (up to 2 mM). Other oximes are very sparingly dispersible in neutral or acidic water in the presence or absence of the metal ions employed, and the concentrations of free or complexed oxime needed for the kinetic measurements (up to the millimolar range) could be obtained by adding cetyltrimethylammonium bromide (CTABr) in at least 5 (1b) or 20 (1c) molar excess. The comicelles made of CTABr and lipophilic oximes turn to metallomicelles by binding the transition metal ions. Complexation is highlighted by the spectral changes observed for pH ) 7 solutions (decrease in the region 240 and 290 nm and increase in (6) Budka, J.; Hampl, F.; Liska, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. J. Mol. Catal., A 1996, 104 (3), 201.

the range 312-330 nm, depending on the metal ion) diagnostic of the formation of complexes. Such spectral changes were instantaneous in the case of Cu(II), Zn(II), and Co(II) and remarkably slow in the case of Ni(II), particularly in acidic solutions; thus, 50% complexation in the case of comicelles of CTABr and 1c (20:1) occurs after 10 min at pH ) 8.5 and after 180 min at pH ) 4.0.7 It is here emphasized that kinetic and any other experiments employing oximes and Ni(II) were carried out after full equilibration was achieved. The cmc value of the comicelles investigated is lower than 0.4 mM (CTABr), and care was taken to employ concentrations well above this value. Direct evaluation of the binding constants of the various complexes and the acid dissociation constants in metallomicelles was not feasible using conventional methods. Indirect information was obtained through the kinetic analysis of the reacting systems. Kinetic Measurements. A preliminary set of experiments was carried out under conditions compatible with the solubility of reagents, using CTABr as the micellar matrix and oxime 1c with and without the divalent ions of Cu, Zn, Co, and Ni, at pH ) 6.3. This pH was chosen to obtain a first indication of the reactivity difference between the uncomplexed (undissociated) and the (at least partly) complexed and dissociated oxime. Some of the results thus obtained are summarized in Table 1 in terms of relative rates (the ratio of pseudo-first-order constants) using PNPH and PNPDPP as substrates. The main information is the following: (i) in the case of the phosphate ester, PNPDPP, the effect of the oxime and all complexes is disappointingly small if any; (ii) in the case of PNPH the kinetic effect strongly depends on the metal ions (upon their addition the reactivity of the oxime was found to decrease in the case of Co(II) and Cu(II) and to increase in the case of Zn(II) and Ni(II)). This preliminary screening and further experiments, which will be partly dealt with below, led us to focus our investigation on the complexes with Zn(II) and particularly with Ni(II) in order to define their mode of action and catalytic efficacy. Reactivity of Oximes 1 in the Presence of Zn(II) and Ni(II). Being bidentate ligands, oximes 1 can form complexes of different ligand/metal ion ratios (stoichiometries). In the case of 1a and Ni(II) complexes, the structures of crystals of 2:1 complexes (L2‚Ni(II)) of different geometries obtained from neutral or mildly acidic solutions were defined as reported.8 Thus, to evaluate the (7) A similar slow complexation of Ni(II) by lipophilic 2-pyridine aldoxime comicellized with CTABr has already been described: Hebrant, M.; Bouraine, A.; Tondre, C.; Brembilla, A.; Lochon, P. Langmuir 1994, 10, 3994. (8) Riggle, K.; Lynde-Kernell, T.; Schlemper, E. O. J. Coord. Chem. 1992, 25, 117.

Metallomicelles Made of Ni(II) Complexes

Figure 1. Kinetic Job plot for the cleavage of PNPH by 1:24 1c/CTABr at pH 6.3 (MES buffer, 0.05 M) in the presence of (]) Ni(II) and (O) Zn(II) and at pH 4.0 (acetate buffer, 0.005 M) in the presence of (9) Ni(II). The sum of the concentrations of ligand and metal ion was kept constant at 0.41 mM. The rate constants obtained at pH ) 4 have been multiplied by 2 to fit the graph.

“effective stoichiometry”, which is not a definition of the nature and quantity of the complexes present in solution but only the most effective ligand/metal ion ratio leading to the highest reactivity observed, we defined the kinetic version of the Job plots shown in Figure 1. These were obtained under the general conditions used for the experiments described above (Table 1) for solutions where [metal ion + oxime 1c] ) 0.41 mM and [CTABr] ) 24 × [1c] and using PNPH as substrate at pH 6.3 (Mes buffer) for Ni(II) and Zn(II) and 4.0 (acetate buffer) for Ni(II). The profiles clearly indicate that in the case of Zn(II) the effective stoichiometry is 1:1 whereas in the case of Ni(II) at pH 4.0 it is 2:1. However, at pH 6.3 and using Ni(II) as metal ion, we obtained a flat maximun in the 0.2-0.3 molar fraction interval which seems to indicate that complexes with 2:1 and 3:1 stoichiometries have similar reactivity. Other Job’s plots for Ni(II) complexes determined at pH 4, in the cases of oxime 1a without CTABr and of oxime 1b and CTABr, again indicate as the most effective stoichiometry a 2:1 ratio. Using the so-estimated oxime/metal ion most effective ratios, we defined the effect of pH for Zn(II) and Ni(II) complexes. As shown in Figure 2, we observed that (i) in the case of the Zn(II) complex the rate linearly increases up to pH ) 8 and then decreases and (ii) in the case of the Ni(II) complex the rate is surprisingly insensitive to pH in a wide pH range (3.5-9). As a result, while at pH ) 8 the (micellar) complex with Zn(II) is more reactive than that with Ni(II) by approximately 1 order of magnitude, at pH ) 6 the reverse is true and, as the acidity of the solution increases, the kinetic benefits observed in the case of the Ni(II) complex, when compared with the other system, are remarkable indeed. Reactivity of Ni(II) Complexes in Acidic Solutions. Figure 3 shows the rate-pH profiles for the cleavage of PNPH in the case of comicelles of the Ni(II) (2:1) mixtures with 1a (no CTABr), 1b (CTABr 10:1), and 1c (CTABr 20:1). Figure 3 also shows the profiles obtained for solutions where Ni(II) is absent, containing 1c (CTABr 20:1) and CTABr only. The main features of these profiles, in most cases encompassing the pH range 2-11, may be summarized as follows: (i) In the case of the comicellized complexes,

Langmuir, Vol. 15, No. 2, 1999 407

Figure 2. pH dependence of the rate constant for the cleavage of PNPH by 1c‚1/2Ni(II)/CTABr (empty symbols) and by 1c‚ Zn(II)/CTABr (filled symbols). Symbols indicate the different buffers used: O, acetate; 0, MES; 4, HEPES; ], EPPS; 3, CHES. Conditions: [Me(II)] ) 0.41 mM; [1c] ) 0.41 mM; [CTABr] ) 1 × 10-2 M in the case of Zn(II) and [1c] ) 0.82 mM; [CTABr] ) 1.6 × 10-2 M in the case of Ni(II).

Figure 3. pH dependence of the rate constant for the cleavage of PNPH by 1a/Ni(II) (0, 2:1), 1b/Ni(II)/CTABr (4, 2:1:20), 1c/ Ni(II)/CTABr (O, 2:1:40), 1c/CTABr (3, 1:20), and CTABr (]). Conditions: [ligand] ) 0.83 mM; [Me(II)] ) 0.41 mM; [buffer] ) 5 mM; for the buffer used, see Figure 2.

on moving from lower to higher pH values, the observed rate increases linearly with a slope close to unity up to a waving plateau extending to pH 11. The change of slope from unity to the plateau region, which can be taken as an indication of the pKa of the effective complexed species, occurs at approximately pH ) 3.7 for both ligands. In the “plateau” region the rate fluctuates within less than 1 order of magnitude. Such fluctuations are similar in shape in the case of 1c and 1b, only somewhat larger in the case of the less lipophilic 1b and larger in the case of PNPH than in that of PNPA (not shown). Not only does the lipophilicity of the ligand and substrate play a role but also the nature and concentration of the buffer employed affect the extent (not so much the shape) of waving of the plateau region. To minimize the effect of the buffer, its concentration was kept rather low (5 mM). (ii) In the case

408 Langmuir, Vol. 15, No. 2, 1999

Hampl et al. Table 2. Kinetic and Thermodynamic Parameters for the Cleavage of PNPH by Comicelles of Different Oxime‚Ni(II) (2:1 Ratio) Complexes and CTABra entry

oxime

[CTABr]/[oxime]

kψlim, s-1

Kb, M-1

kψlim/k0b

1 2 3 4

1b 1b 1b 1c

5 10 20 20

0.15 0.077 0.025 0.010

4,250 1,760 1,270 3,610

508,500 261,000 84,700 33,900

a Conditions: 0.005 M acetate buffer, pH 4; 25 °C. b k ) 2.95 × 0 10-7 s-1; rate constant in pure buffer.

Figure 4. Rate constant versus 1b concentration profiles for the cleavage of PNPH in acetate buffer at pH 4 at 25 °C and in the presence of Ni(II) and CTABr. 1b/Ni(II)/CTABr ratio: (O) 2:1:40; (0) 2:1:20; (4) 2:1:10.

of the nonmicellized complex of 1a, the rate increases linearly with slope ) 1 up to pH 5 and, afterward, with a smaller slope (ca. 0.3-0.4) up to pH 11. In this case, the rate increases with pH in such a way that, although at pH ) 3 the nonmicellized complex is approximately 2.5 orders of magnitude less effective than the comicellized analogue with 1c, above pH ) 8 the reactivities of the micellized and not micellized oximes in the presence of Ni(II) are virtually similar. (iii) In the case of micellized 1c, in the absence of the metal ion, the reactivity at low pH is more than 3 orders of magnitude lower than that of the corresponding comicellized complex and then increases, albeit with a slow start, up to the point (pH > 8) when its reactivity exceeds that of any other system considered. (iv) In a solution of CTABr only, the rate of hydrolysis, after a plateau region in the pH range 2-4, increases linearly with a slope of 1 and is constantly approximately 1 order of magnitude lower than that of the uncomplexed micellized 1c. The data of Figure 3 clearly indicate that the kinetic benefits of the ketoximes are mainly confined to the acidic region, where the reactivity of the Ni(II) complexes is remarkably larger than that of free oximes and is consistently magnified by micellization. A more quantitative evaluation of the rate acceleration at pH 4 was obtained from measurements carried out using solutions containing increasing amounts of 1b (in each case with Ni(II) in the molar ratio 2:1) and CTABr with molar ratios, relative to the oxime, of 20, 10, and 5, using PNPH as substrate: the resulting rate-concentration profiles are shown in Figure 4. Analogous profiles were also obtained with comicelles of CTABr and 1c in a 20:1 ratio. Analysis9 of the curves, showing a saturation behavior, allows us to estimate the apparent binding constants (Kb) for PNPH in the different comicelles and the kψlim, that is, the rate constants expected for the PNPH being totally incorporated into the aggregates. These are reported in

Table 2. Somewhat unexpectedly, in the case of 1b the apparent Kb increases as the ratio CTABr/oxime decreases and for a 20:1 ratio the Kb value is greater for 1c than for 1b. On the other hand, on decreasing the surfactant/ligand ratios, the kψlim values substantially increase. The maximum kinetic benefit is that evaluated for the CTABr/1b ) 5 mixture: the limiting value amounts to over half million-fold that measured in the absence of oxime and of Ni(II). Analogous profiles were determined for comicelles of CTABr/1b ) 5, but in the absence of Ni(II): in such a case, above the cmc, the rate increases (albeit to a small extent) to reach a plateau value at a concentration approximately six times lower than that observed in the presence of the metal ion. These results indicate that the apparent Kb evaluated for the micellized metallocomplexes comprises, besides binding of the substrate to the comicelles, also binding of the complexed ligands; moreover, it is certainly affected by the fact that the constants for metal ion binding to the oxime are relatively small (see below). In view of the latter point, the values of Table 2 can be taken as the lower limits of the rates of cleavage of the ester, since not all the metal ion is complexed to the oxime. Oxime 1a and its methylated analogue 2a are sufficiently soluble in water to enable us to investigate their reactivities in the absence of CTABr. The kψ versus [ligand] profile obtained using a 2:1 mixture of ligand and Ni(II) and PNPH as substrate at pH ) 4.0 describes straight lines with a slope of 0.07 in the case of 1a and a slope close to zero with 2a. At [ligand] ) 1.1 mM the 1a-Ni mixture is 80-fold more reactive than that of 2a and Ni and 260 times over that in pure buffer. Virtually the same type of profile was obtained using the more hydrophilic PNPA as a substrate. Mode of Action. The inertness of the Ni(II) complex with 2a is a clear indication that, at least in the case of non-comicellized systems, the oxime hydroxyl is involved as a nucleophile in the cleavage of the carboxylic esters, and it is quite reasonable to assume this function is acylated to give an intermediate which in turn may be deacylated to restore the catalytic complex and define a catalytic cycle. To prove such an hypothesis, we carried out a set of experiments using comicellized complexes of 1b and 1c using PNPH and PNPA as substrates in a molar excess (concentrations 7-10 times larger than that of the catalyst). “Burst” kinetics such as those shown in Figure 5 were observed, indicating that the p-nitrophenol is first released in a fast (“burst”, kinetically first-order) process in an amount equal to that of the ligand and then in a slower (zeroth-order) process. In the case illustrated in Figure 5, from the slope of the linear tract after the “burst” and the  value evaluated under the conditions used, an apparent (underestimated, (9) The kψ versus [oxime] data were fitted with the Michaelis-Menten equation. Using the same equation and as the concentration of catalyst the total concentration of surfactant, or another equation currently used in the analysis of micellar systems,5c, d we obtained identical values of kψlim and values of Kb which follow the same trend.

Metallomicelles Made of Ni(II) Complexes

Langmuir, Vol. 15, No. 2, 1999 409 Table 3. Rate Constants of Acylation (ka, M-1 s-1) and Deacylation (kd, s-1) for Different Oxime‚Ni(II) (2:1 Ratio) Complexes in the Cleavage of PNPAa oxime

ka (M-1 s-1)

kd (s-1)

1a 1bb

0.8 0.3

1.0 × 10-3 1.4 × 10-4

a Conditions: 0.05 M acetate buffer, pH 4; 25 °C. b In the presence of a 10 fold excess of CTABr.

Figure 5. Time course for the release of p-nitrophenol in the cleavage of PNPA by 1b‚Ni(II)/CTABr in the presence of an excess of substrate over catalyst. Conditions: [PNPA] ) 0.45 mM; [1b] ) 0.06 mM; [Ni(II)] ) 0.03 mM; [CTABr] ) 0.54 mM; 0.05 M acetate buffer, pH 4; 25 °C; λ ) 317 nm.

see below) turnover rate of 0.37 × 10-4 s-1 may be evaluated, assuming that the concentration of catalyst is equal to that of the complex ligand2‚Ni(II). The turnover is appreciably large to justify the definition of catalyst for the system under study. The behavior apparent in Figure 5 fits nicely a mechanistic scheme involving nucleophilic attack by the oxime ligand on the ester and acylation of all the available ligand, followed by a slower process controlled by the deacylation of the ligand: this implies the formation of a relatively stable intermediate. To define the formation and structure of such an intermediate, the reaction was carried out under conditions of excess PNPA, as in the kinetic experiments described above, and just after the “burst” phase, the process was quenched by adding an excess of EDTA. Extraction of the organic material from the solution containing 1b as ligand allowed the isolation of the O-acylated ligand, 2c, which turned out to be identical to the compound independently synthesized. The same type of intermediate (2b) was isolated and identified using the nonmicellized Ni(II) mixture of 1a. The “burst” kinetics were particularly well behaved in the case of PNPA, which is more soluble than PNPH, to allow a sufficiently large molar excess, still maintaining the concentration of the comicelles above the cmc. These experiments led to a reasonable evaluation, by means of a described treatment of the data,5d,10 of the rate of formation of the acylated intermediate, ka, and that of its deacylation, kd, under the conditions employed. It is here emphasized that the main source of uncertainty in evaluating the second-order rate constants and the rate of the turnover is the evaluation of the fraction of complexed oxime, that is, of the effective catalyst concentration, particularly in comicelles. This was estimated by using the pKNi value reported for the 2-pyridinealdoxime,4a clearly a gross approximation. The kinetic constants thus evaluated are reported in Table 3. Ni(II)-Catalyzed Hydrolysis of the Acetylated Ketoximes. The availability of the acetylated ketoximes 2b,c turned our attention to their hydrolytic behavior in the presence of Ni(II) and, in the case of the more lipophilic 2c, of comicelles with CTABr. Taking into account the extensive work by Suh11 on the mechanisms of the Cu(II)-, (10) Suh, J.; Cheong, M.; Han, H. Bioorg. Chem. 1984, 12, 188.

Figure 6. Kinetic Job plot for the cleavage of 2b by Ni(II) at pH 4 (acetate buffer, 0.05 M).

Zn(II)-, and Ni(II)-catalyzed hydrolysis of a variety of O-acetylated pyridineoximes, this side investigation was mainly performed to compare the rate of hydrolysis of the acetylated terms with those obtained from the burst kinetics, as a further evidence of their intermediacy in the catalytic reaction of the corresponding ketoximes, and to define the role of the cationic aggregates in the process. In a first set of kinetic experiments, we tried to define the effective stoichiometry of the complexed species by means of the kinetic versions of the Job plot. As shown in Figure 6 in the case of 2b and as found in the case of 2c (not shown) the best ratio is 1:1. In a second set of experiments we measured the rate of deacylation of 2b and 2c by keeping constant the concentration of the ligand (and CTABr in the case of 2c) and increasing that of Ni(II) ion. With both ligands, saturation curves such as that shown in Figure 7 were obtained and analyzed as described below. Under the assumption that (i) the mode of action involves nucleophilic attack by an activated, metal-bound water molecule at the ester linkage, as established by Suh12 for analogous systems, and (ii) the effective stoichiometry of the complex is 1:1, application of the kinetic law12

kψ )

kd[Ni(II)] (1/K + [Ni(II)])

where kψ is the observed rate constant, kd is the deacylation (first order) rate constant, and K is the apparent com(11) (a) Suh, J. Bioorg. Chem. 1990, 18, 345. (b) Suh, J. Acc. Chem. Res. 1992, 25, 274 and references therein. (12) Suh, J.; Chang, B. Bioorg. Chem. 1987, 15, 167.

410 Langmuir, Vol. 15, No. 2, 1999

Hampl et al.

Figure 7. Rate constant versus Ni(II) concentration profiles for the cleavage of 2c in the presence of CTABr at pH 4. Conditions: [2c] ) 0.1 mM; [CTABr] ) 0.5 mM; 0.05 M acetate buffer, pH ) 4; T ) 25 °C. Table 4. Apparent Deacylation (kd, s-1) and Metal Ion Binding (K, M-1) Constants for 2b,c and Ni(II) in 0.05 M Acetate Buffer at pH 4 at 25 °C ligand

[CTABr]/[ligand]

kd, s-1

K, M-1

2b 2c 2c 2c

5 10 20

1.2 × 10-3 9.0 × 10-4 9.5 × 10-4 1.1 × 10-3

950 140 48 20

plexation constant of the free ligand in the case of 2b and of the micellized ligand (with CTABr in molar excesses ranging from 5 to 20) in that of 2c, leads to the values of Table 4. When compared to the deacylation rate constants obtained from the burst experiments, although performed employing a 2:1 ligand/Ni(II) ratio (see Table 3), that of 2b is virtually similar while the value for the comicellized 2c is 6-7 times higher and virtually independent of the molar excess of CTABr. On the other hand, the Ni(II) binding constant for 2b is very similar to that reported by Suh and co-workers,12 whereas in the case of 2c the value is lower than that of the free analogue and decreases with the increasing CTABr/ligand ratio.13 Discussion The most interesting results of the present study are illustrated in Figure 3, showing the kinetic data obtained for the hydrolysis of activated carboxylic acid esters in acidic aqueous solutions. The large acceleration (up to more than 5 orders of magnitude relative to pure buffer, as reported in Table 2) observed in the case of the comicellized complexes of pyridineketoximes 1b,c with Ni(II) is quite impressive when one takes into account the low pH range (3-5) and the mode of action involving a nucleophilic attack by the oxime function at the carbonyl carbon of the ester. Accelerations of over 6 orders of magnitude were reported in the hydrolysis of R-amino acid esters employing complexes of OH-functionalized ligands with Cu(II);1c however, in that case the mode of action is quite different, as it involves a ternary (ligand/ metal ion/substrate) complex where the substrate is associated through its free amino group to the Cu(II) ion acting as a template. In the present case the carboxylate esters are not bound to the ion and only brought into the proximity of the nucleophilic species mainly by hydro(13) A decrease of the formation constant by dissolving the ligand in cationic micelles is expected: Scrimin, P.; Tecilla, P.; Tonellato, U.; Vendrame, T. J. Org. Chem. 1989, 54, 5988.

Figure 8. Proposed catalytic cycle for the cleavage of carboxylic esters by oxime2‚Ni(II) complexes at pH 4.

phobic forces when included into the small volume of the micellar aggregates. The reaction mechanism as based on the results here reported can be reasonably defined by the catalytic cycle of Figure 8 operative at pH values around 4. The effective 2:1 complex A, only partly dissociated (see below), cleaves the ester following nucleophilic attack of the oximate function to give the complexed acylated species (2b or 2c are formed from 1a and 1b, respectively) and p-nitrophenol. The acylated complex is then hydrolyzed by the action of an activated water molecule bound to the complex, and the system turns over. After acylation, the structure or conformation of the complex may change: so, one ligand may move out to form a 1:1 complex that is the most effective species for the deacylation process, as established from the present studies and from published data.12 However, as indicated by the Job plot of Figure 6, a 2:1 complex, as is tentatively represented as B in Figure 8, is probably still effective and is here suggested as the reasonable intermediate in micellar systems where the complexation and, probably, the decomplexation process are slow (see above) in the time scale of the whole catalytic cycle. On moving from pH 4 to neutral or mildly basic solutions, the reactivity of the comicellar systems containing the oxime complexes with Ni(II) first slightly decreases and then remains virtually constant, so that it approaches that of the comicellar uncomplexed oxime around pH 8. On the other hand, the reactivity of the free (comicellized) oxime regularly increases from pH 8 to 11, as expected from the deprotonation of the oxime with a pKa of approximately 12. Thus, the metal ion decreases the apparent pKa of the oxime outside the aggregates (see 1a) by almost 7 orders of magnitude and by 8-9 orders of magnitude when residing in cationic (CTABr) micelles. The decrease in the pKa of the complexed oxime is larger for Ni(II) than for Zn(II) (see Figure 2), although from the literature it is probably lower than that with Cu(II).4b Therefore, the presence of the oximate complexed species even in neutral or slightly acidic conditions is not surprising; although bound to the metal ion, it is a much more effective nucleophile than the complexed undissociated oxime. However, in the present study of the Ni(II) complexes of the 2-pyridineketoxime, there are some peculiar and intriguing effects which may be summarized as follows: (i) the 2:1 stoichiometry which is extremely efficient toward the CdO group of the carboxylic acid esters in an unusual

Metallomicelles Made of Ni(II) Complexes

pH range but disappointingly inefficient toward the PdO group of phosphoric acid esters; (ii) the apparent deactivation of the fully dissociated oximate functions as nucleophiles when bound to the metal ion. As for point i, our rationale is as follows. Complex A is drawn in Figure 8 in such a conformation where the four nitrogen atoms of the two ligand molecules surround the metal ion in a planar arrangement and the two oxime functions face each other (in a cis fashion): one neutral and the other dissociated, kept together by an hydrogen bond. As a matter of fact, this is the structure of the 2:1 complex of 1a with Ni(II) crystallized from aqueous solutions of pH 5, as reported by Riggle et al.8 Although one can hardly take for granted that this is the actual conformation in solution, it appears as a reasonably stabilized anionic species also at low pH. It is here noteworthy that the crystal structure of the same complex isolated from a solution at pH 3 shows that the undissociated oxime functions are located on the opposite side (trans fashion). Assuming that electrostatic factors play a role in favoring the trans arrangement, the dioximate complex present at higher pH values should also assume this configuration. Thus the compact structure of species A is probably confined in a relatively narrow pH range and is a better nucleophilic species, at least in micelles, than the undissociated and the doubly dissociated (neutral) complexes active at higher pHs. Although other factors may play a role in enhancing its reactivity, the main effect appears to be due to the micellar ambient. In the micellar pseudophase the two paraffinic tails of complex A, in the cis conformation, are more easily aligned than those in the trans one, and this may favor a deeper insertion and a better juxtaposition of the nucleophilic site with the ester. Conformation A should also favor a possible cooperativity between the two oximic functions: the intermediate and the transition state resulting from the nucleophilic attack of the oximate on the carbonyl carbon could be stabilized by the proton, which, at any rate, could assist the departure of the leaving group. Point ii, the apparent deactivating effect of the Ni(II) bound to the oximate, is also supported by the evidence reported by two of us (F.H. and F.L.) from a study of the hydrolytic efficacy of metallomicelles made of or containing cationic surfactants derived from pyrineketoxime.14 In such a case, the transition metal ions investigated, Co(II), Cu(II), and Ni(II), were found to decrease the nucleophilicity of the oximate functions to a larger extent than that observed in the present study. This effect may be general, related to the lowering of the pKa and hence of the basicity and nucleophilicity of the oxime function, or may be a specific feature of the complex between the oxime and the metal ion. Indeed the NdO- group, as an R-nucleophile, is a special case, the nitrogen atom being the donor for metal ion binding and the oxygen the actual nucleophilic site. Binding involves depletion of the available electrons at the nitrogen, and on this ground one may expect a decreased R-nucleophilicity of the neighboring oxygen. This is an interesting point which, however, requires further experiments particularly addressed to the correct evaluation and comparison of the second-order rate constants for the reaction of the free and the complexed oximes. Finally the scarce reactivity of the complexes of the oxime investigated toward phosphate esters vis-a`-vis that toward carboxylate esters is a matter of disappointment in view of our premises based on the efficacy of PAM,2 at least in vivo, and of the lipophilic analogue of PAM in cationic micelles.15 Further systematic investigations are in progress, aimed at substantiating the present results

Langmuir, Vol. 15, No. 2, 1999 411

and possibly understanding the mechanistic aspects of the system involving the phosphate derivatives. Conclusion The rate enhancements of over 5 orders of magnitude in the cleavage of carboxylic acid esters in mildly acidic solutions are unprecedent for any functionalized metallomicellar system so far reported at any pH value. Only in one case using homomicelles made of complexes of the ligand 6-((n-dodecylamino)methyl)-2-(hydroxymethyl)pyridine with Cu(II) did we observed1g a remarkable reactivity (limiting accelerations of over 3 orders of magnitude) in the cleavage of PNPH in neutral (pH ) 6.25) solutions. Interestingly, in both cases, a ligand to metal 2:1 stoichiometry was the most effective one. In the case of the Cu(II) metallomicelles, the nucleophilic functions were indicated as the (at least partly) dissociated hydroxy function probably not too tightly bound to the metal ion and kept in a rather flexible position due to the presence of the several competing chelating sites in the 2:1 complex, and the overall efficiency was attributed to the concentration effect in the aggregates. The availability of an activated but not (or weakly) coordinated Onucleophilic site is apparently the common feature for the two efficient micellar systems in the cleavage of noncoordinating substrates such as carboxylic acid esters. However, there is an intriguing difference which is related to the phosphate esters: whereas the above Cu(II) metallomicelles are effective catalysts in the cleavage of phosphate esters such as PNPDPP, the present Ni(II) system is not. One may speculate that the oximate function kept close to the crowded complex (A of Figure 8) by strong binding does not interact for steric reasons with the PdO center as the hydroxy function in the Cu(II) complex does. Of course this is an interesting point which deserves further study in view also of the premises based on the effectiveness of PAM. Experimental Section General Methods and Materials. Melting points are uncorrected. 1H NMR spectra were recorded on Bruker AC 250F and Bruker AM 400 spectrometers operating at 250 and 400 MHz, and chemical shifts are reported relative to internal Me4Si. Elemental analyses were performed by the Laboratorio di Microanalisi of the Organic Chemistry Department of the University of Padova and by the Laboratory of Organic Analysis of the ICT. Surface tension measurements were performed with a Kruss type 8451 tensiometer. UV-vis spectra and kinetic traces were recorded on a Perkin-Elmer Lambda 16 spectrophotometer equipped with a thermostated cell holder. Cu(NO3)2, Zn(NO3)2, Ni(NO3)2, and CoCl2 were analytical grade products. Metal ion stock solutions were titrated against EDTA following standard procedures.16 The buffer components were used as supplied by the manufacturers: acetic acid (Aldrich); 2-morpholinoethanesulfonic acid (MES, Fluka); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma); 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS, Aldrich); 2-(cyclohexylamino)ethanesulfonic acid (CHES, Aldrich); 3-(cyclohexylamino)-1propanesulfonic acid (CAPS, Aldrich). n-Hexadecyltrimethylammonium bromide (CTABr, Fluka) was an analytical grade commercial product. The p-nitrophenyl esters of acetic (PNPA) and hexanoic acid (PNPH) were Sigma products used as received. The p-nitrophenyl esters of diphenylphosphoric acid (PNPDPP) (14) Cibulka, R.; Dvora´k, D.; Hampl, F.; Liska, F. Collect. Czech. Chem. Commun. 1997, 62, 1342. (15) (a) Epstein, J.; Kaminski, J. J.; Bodor, N.; Enever, R.; Sowa, J.; Higuchi, T. J. Org. Chem. 1978, 43, 2816. (b) Reiner, R.; Rossman, K. Monatsh. Chem. 1982, 113, 223. (c) Lion, C.; Despagne, B.; Delmas, G.; Fosset, L. Bull. Soc. Chim. Belg. 1991, 100, 549. (d) Hampl, F.; Mazac, J.; Liska, F.; Srogl, J.; Kabrt, L.; Suchanek, M. Collect. Czech. Chem. Commun. 1995, 60, 883. (16) Holzbecher, Z. Handbook of Organic Reagents in Inorganic Analysis; Wiley: Chichester, 1976.

412 Langmuir, Vol. 15, No. 2, 1999 were prepared by literature methods.17 The syntheses of 1-(2pyridinyl)ethanone oxime (1a) and 1-(2-pyridinyl)ethanone Oacetyloxime (2b) were performed as reported.18 1-(2-Pyridinyl)ethanone O-Methyloxime (2a). To a solution of 1-(2-pyridinyl)ethanone (0.56 g, 4.1 mmol) in 5 mL of EtOH were added first a solution of methoxyamine hydrochloride (0.51 g, 6.1 mmol) in 5 mL of EtOH and then a solution of K2CO3 (0.4 g, 2.9 mmol) in 5 mL of water. The reaction mixture was stirred at room temperature for 4 h and then refluxed for 2 h. After addition of 4 mL of water, the reaction mixture was extracted with CHCl3 (3 × 8 mL) and the dried (Na2SO4) organic solution was evaporated to leave a crude material which was purified by column chromatography (silica gel, CHCl3/CH3OH 100:2), affording 0.42 g (68%) of pure 2a as a clear oil. 1H NMR (CDCl3): 2.33 (s, 3 H, CH3); 4.01 (s, 3 H, OCH3); 7.16 (ddd, J ) 1.2, 4.9, and 7.5 Hz, 1 H, H5Py); 7.59 (dt, J ) 1.8 and 7.8 Hz, 1 H, H4Py); 7.88 (dd, J ) 1.1 and 8 Hz, 1 H, H3Py); 8.56 (ddd, 1 H, J ) 0.9, 1.7, and 4.9 Hz, H6Py). Anal. Calcd for C8H10N2O: C, 63.98; H, 6.71. Found: C, 64.14; H, 7.10. 1-(2-Pyridinyl)-1-nonanone Oxime (1b). To a solution of the Grignard reagent prepared from 1.1 g of Mg and octylbromide (6.44 g, 0.033 mol) in 75 mL of dry Et2O was added drop by drop a solution of 2-cyanopyridine (3.1 g, 0.03 mmol) in 100 mL of dry Et2O. After the reaction mixture was stirred for one night at room temperature, 50 mL of water was added. The aqueous layer was made acidic with H2SO4, and the two phase were separated. Evaporation of the dried (Na2SO4) organic solvent afforded a crude which was purified by column chromatography (silica gel, CHCl3/CH3OH 100:1), giving 3.29 g (50%) of 1-(2-pyridinyl)-1nonanone as a yellowish oil. 1H NMR (CDCl3): 0.87 (m, 3 H, (CH2)5CH3); 1.3 (m, 10 H, (CH2)5CH3); 1.73 (m, 2 H, C(dO)CH2CH2); 3.21 (t, J ) 7.3 Hz, 2 H, C(dO)CH2); 7.43 (ddd, J ) 1.1, 4.8, and 7.5 Hz, 1 H, H5Py); 7.83 (dt, J ) 1.8 and 7.7 Hz, 1 H, H4Py); 8.04 (dd, J ) 1.1 and 8 Hz, 1 H, H3Py); 8.68 (m, 1 H, H6Py). To a solution of the previous compound (1.5 g, 6.8 mmol) in 10 mL of EtOH were added first a solution of hydroxylamine hydrochloride (0.6 g, 8.5 mmol) in 5 mL of water and then 0.6 g of Na2CO3 dissolved in 6 mL of water. The reaction mixture was stirred and heated at 60 °C for 2 h. Water (30 mL) was added, and the aqueous phase was extracted with CHCl3 (3 × 50 mL). The organic phase was dried (Na2SO4), and the solvent was evaporated, leaving a crude material which was purified by column chromatography (silica gel, CHCl3/CH3OH 100:2), yielding 1.4 g (88%) of pure 1-(2-pyridinyl)-1-nonanone oxime (1b) as a white solid, mp 38-40 °C. 1H NMR (CDCl3): 0.86 (t, J ) 6.4 Hz, 3 H, (CH2)5CH3); 1.3 (m, 10 H, (CH2)5CH3); 1.57 (m, 2 H, C(dN)CH2CH2); 2.97 (t, J ) 7.5 Hz, 2 H, C(dN)CH2); 7.25 (ddd, J ) 1.2, 4.8, and 7.4 Hz, 1 H, H5Py); 7.67 (dt, J ) 1.8 and 7.5 Hz, 1 H, H4Py); 7.80 (dd, J ) 1.1 and 8 Hz, 1 H, H3Py); 7.94 (bs, 1 H, NOH); 8.61 (m, 1 H, H6Py). Anal. Calcd for C14H22N2O: C, 71.76; H, 9.46; N, 11.95. Found: C, 71.74; H, 9.44; N, 11.98. 1-(2-Pyridinyl)-1-nonanone O-Acetyloxime (2c). 1-(2Pyridinyl)-1-nonanone oxime (0.3 g, 1.3 mmol) and 3 mL of acetic anhydride were stirred and heated at 80 °C for 2 h. After the mixture was cooled at room temperature, a 5% solution of Na2CO3 was added to the reaction mixture to bring the pH to 8. The water phase was extracted with CHCl3 (3 × 30 mL), and the evaporation of the dried (Na2SO4) organic solvent afforded a crude material which was purified by column chromatography (silica gel, toluene/ethyle acetate 7:3), yielding 0.2 g (56%) of pure 2c as an oily product. 1H NMR (CDCl3): 0.87 (t, J ) 6.4 Hz, 3 H, (CH2)5CH3); 1.3 (m, 10 H, (CH2)5CH3); 1.57 (m, 2 H, C(dN)CH2CH2); 2.72 (s, 3 H, OC(O)CH3); 3.04 (t, J ) 7.5 Hz, 2 H, C(dN)CH2); 7.32 (ddd, J ) 1.2, 4.8, and 7.4 Hz, 1 H, H5Py); 7.71 (dt, J ) 1.8 and 7.7 Hz, 1 H, H4Py); 8.02 (dd, J ) 1.1 and 8.0 Hz, 1 H, H3Py); 8.64 (m, 1 H, H6Py). Anal. Calcd for C16H24N2O2: C, 69.53; H, 8.75; N, 10.14. Found: C, 69.22; H, 8.71; N, 10.02. (17) Gulick, W. M., Jr.; Geske, D. H. J. Am. Chem. Soc. 1966, 88, 2928. (18) Blanch, J. H.; Onsanger, O. T. J. Chem. Soc. 1965, 3737. (19) Menasse`, R.; Klein, G.; Erlenmeyer, H. Helv. Chim. Acta 1955, 38, 1289. (20) The fit of the data was made using the software package Enzfitter: Leatherbarrow, R. J. Enzfitter; Elsevier: Amsterdam, 1987.

Hampl et al. 1-(2-Pyridinyl)-1-tetradecanone Oxime (1c). To a supension of sodium powder (12.8 g, 0.557 mol) in 600 mL of dry Et2O was added a mixture of ethyl acetate (50 g, 0.568 mol) and ethyl picolinate (76 g, 0.502 mol). The solution was stirred and refluxed under nitrogen for 2 h. During this time a brownish-yellow precipitate was formed. After the mixture was cooled to room temperature, the precipitate was collected by filtration, washed with a small amount of cold Et2O, and dried to give 74.2 g (68%) of the sodium salt of 3-oxo-3-(2-pryridyl)propanoic acid ethyl ester as a solid, mp 226-229 °C.19 To a solution of the previous material (10 g, 0.046 mol) in 200 mL of dry DMF was added dodecyl bromide (11.6 g, 0.046 mol). The reaction mixture was stirred and heated at 140 °C for 4 h. The solvent was removed under vacuum, and the residue was treated with 300 mL of Et2O. A white precipitate of NaBr was filtered off, and the organic solvent was evaporated. To the material thus obtained was added 150 mL of a 20% water solution of hydrochloric acid, and the resulting mixture was heated at reflux until the evolution of carbon dioxide was completed (about 4 h). After neutralization with solid Na2CO3, the water was extracted with chloroform (4 × 50 mL). The organic solvent was dried (Na2SO4) and evaporated to leave a crude material which was purified by column chromatography (silica gel, CHCl3/CH3OH 100:1), yielding 5.52 g (41%) of 1-(2-pyridinyl)-1-tetradecanone. An analytical sample was obtained by sublimation, mp 28-31 °C. 1H NMR (CDCl3): 0.88 (t, J ) 6.8 Hz, 3 H, (CH2)12CH3); 1.32 (m, 20 H, (CH2)10CH3); 1.73 (m, 2 H, C(dO)CH2CH2); 3.21 (t, J ) 7.5 Hz, 2 H, C(dO)CH2); 7.44 (dd, J ) 4.8 and 7.6 Hz, 1 H, H5Py); 7.82 (t, J ) 7.8 Hz, 1 H, H4Py); 8.04 (d, J ) 7.8 Hz, 1 H, H3Py); 8.67 (d, J ) 4.8 Hz, 1 H, H6Py). Anal. Calcd for C19H31NO: C, 78.84; H, 10.79; N, 4.84. Found: C, 78.94; H, 10.75; N, 4.78. To a solution of the previous ketone (4.05 g, 14 mmol) in 50 mL of EtOH were added two saturated water solutions of hydroxylamine hydrochloride (1.22 g, 18 mmol) and Na2CO3 (1.21 g, 9 mmol). The reaction mixture was stirred and heated at 60 °C for 3 h. Water (30 mL) was added, and the aqueos phase was extracted with CHCl3 (3 × 50 mL). The organic phase was dried (Na2SO4), and the solvent was evaporated, leaving a crude material which was purified by column chromatography (silica gel, CHCl3/CH3OH 100:2), yielding 3.36 g (79%) of pure 1-(2pyridinyl)-1-tetradecanone oxime (1c) as a white solid, mp 6970 °C. 1H NMR (CDCl3): 0.89 (t, J ) 6.8 Hz, 3 H, (CH2)12CH3); 1.35 (m, 20 H, (CH2)10CH3); 1.62 (m, 2 H, C(dN)CH2CH2); 2.98 (t, J ) 7.2 Hz, 2 H, C(dN)CH2); 7.25 (ddd, J ) 1.3, 4.7, and 7.4 Hz, 1 H, H5Py); 7.74 (dt, J ) 1.7 and 7.4 Hz, 1 H, H4Py); 7.84 (dd, J ) 1.2 and 8 Hz, 1 H, H3Py); 8.68 (m, 1 H, H6Py); 8.86 (bs, 1 H, NOH). Anal. Calcd for C19H32N2O: C, 74.95; H, 10.59; N, 9.20. Found: C, 74.95; H, 10.51; N, 9.09. Kinetic Studies. The reactions were followed on a PerkinElmer Lambda 16 spectrophotometer equipped with a thermostated cell holder. Solutions of the ligands, metal ions, and additives were prepared in the proper buffer. The reaction temperature was maintained at 25 ( 1 °C. Reactions were started by addition of 20 µL of a (1-2) × 10-3 M solution of substrate in CH3CN to 2 mL of a solution of ligand and additives, and no changes in pH were observed during the kinetic runs. The final concentration of substrate was (1-2) × 10-5 M, and the kinetics are in each case first-order up to 90% of reaction. The rate constants were obtained by nonlinear regression analysis of the absorbance versus time data,20 and the fit error on the rate constant was always less than 1%. To measure the pH dependence of the hydrolysis rate, the following buffers were used: acetate, 4.0 < pH < 5.3; MES, 5.3 < pH < 6.7; HEPES, 6.7 < pH < 7.7; EPPS, 7.7 < pH < 8.7; CHES, 8.7 < pH < 9.7; CAPS, 9.6 < pH < 10.6.

Acknowledgment. Financial support for this research has been provided by the Ministry of the University and Scientific and Technological Research (MURST) and by the Grant Agency of the Czech Republic (grant nos. 203/ 93/0546 and 203/97/0805). The authors thank Dr. G. Santacatterina for her participation and Mr. E. Castiglione for technical assistance. LA980861+